Isothermal amplification components and processes

ABSTRACT

The technology relates in part to methods and compositions for isothermal amplification of nucleic acids.

RELATED PATENT APPLICATIONS

This patent application is a 35 U.S.C. 371 national phase application ofInternational Patent Cooperation Treaty (PCT) Application No.PCT/US2017/020921, filed on Mar. 6, 2017, entitled ISOTHERMALAMPLIFICATION COMPONENTS AND PROCESSES, naming Andrew P. Miller andHonghua Zhang as inventors, which claims the benefit of U.S. patentapplication Ser. No. 15/090,405 filed on Apr. 4, 2016, entitledISOTHERMAL AMPLIFICATION COMPONENTS AND PROCESSES, naming Andrew P.Miller and Honghua Zhang as inventors. The entire content of theforegoing application is incorporated herein by reference, including alltext, tables and drawings, for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 2, 2017, isnamed NAT-1001-PC_SL.txt and is 16,533 bytes in size.

FIELD

The technology relates in part to methods and compositions forisothermal amplification of nucleic acids.

BACKGROUND

Nucleic acid-based diagnostics can be useful for rapid detection ofinfection, disease and/or genetic variations. For example,identification of bacterial or viral nucleic acid in a sample can beuseful for diagnosing a particular type of infection. Other examplesinclude identification of single nucleotide polymorphisms for diseasemanagement or forensics, and identification of genetic variationsindicative of genetically modified food products. Often, nucleicacid-based diagnostic assays require amplification of a specific portionof nucleic acid in a sample. A common technique for nucleic acidamplification is the polymerase chain reaction (PCR). This techniquetypically requires a cycling of temperatures (i.e., thermocycling) toproceed through the steps of denaturation (i.e., separation of thestrands in the double-stranded DNA (dsDNA) complex), annealing ofoligonucleotide primers (short strands of complementary DNA sequences),and extension of the primer along a complementary target by apolymerase. Such thermocycling can be a time consuming process thatgenerally requires specialized machinery. Thus, a need exists forquicker nucleic acid amplification methods that can be performed withoutthermocycling. Such methods may be useful, for example, for on-sitetesting and point-of-care diagnostics.

SUMMARY

Provided herein in certain aspects are methods for amplifying nucleicacid, comprising contacting non-denatured sample nucleic acid underisothermal amplification conditions with components comprising a) atleast one oligonucleotide, which at least one oligonucleotide comprisesa polynucleotide complementary to a target sequence in the samplenucleic acid, and b) at least one component providing hyperthermophilepolymerase activity, thereby generating a nucleic acid amplificationproduct.

Also provided herein in certain aspects are methods for amplifyingnucleic acid, comprising contacting non-denatured sample nucleic acidunder isothermal amplification conditions with a) non-enzymaticcomponents comprising at least one oligonucleotide, which at least oneoligonucleotide comprises a polynucleotide complementary to a targetsequence in the sample nucleic acid, and b) an enzymatic componentconsisting of a hyperthermophile polymerase or a polymerase comprisingan amino acid sequence that is at least about 90% identical to ahyperthermophile polymerase, thereby generating a nucleic acidamplification product.

Also provided herein in certain aspects are methods for amplifyingnucleic acid, comprising contacting non-denatured sample nucleic acidunder isothermal amplification conditions with a) non-enzymaticcomponents comprising at least one oligonucleotide, which at least oneoligonucleotide comprises a polynucleotide complementary to a targetsequence in the sample nucleic acid, and b) enzymatic activityconsisting of i) hyperthermophile polymerase activity and, optionally,ii) reverse transcriptase activity, thereby generating a nucleic acidamplification product.

Also provided herein in certain aspects are methods for processingnucleic acid, comprising amplifying nucleic acid, where the amplifyingconsists essentially of contacting non-denatured sample nucleic acidunder isothermal amplification conditions with a) at least oneoligonucleotide, which at least one oligonucleotide comprises apolynucleotide complementary to a target sequence in the sample nucleicacid, and b) at least one component providing hyperthermophilepolymerase activity, thereby generating a nucleic acid amplificationproduct.

Also provided herein in certain aspects are methods for processingnucleic acid, comprising amplifying nucleic acid, where the amplifyingconsists essentially of contacting non-denatured sample nucleic acidunder isothermal amplification conditions with a) non-enzymaticcomponents comprising at least one oligonucleotide, which at least oneoligonucleotide comprises a polynucleotide complementary to a targetsequence in the sample nucleic acid, and b) an enzymatic componentconsisting of a hyperthermophile polymerase or a polymerase comprisingan amino acid sequence that is at least about 90% identical to ahyperthermophile polymerase, thereby generating a nucleic acidamplification product.

Also provided herein in certain aspects are methods for processingnucleic acid, comprising amplifying nucleic acid, where the amplifyingconsists essentially of contacting non-denatured sample nucleic acidunder isothermal amplification conditions with a) non-enzymaticcomponents comprising at least one oligonucleotide, which at least oneoligonucleotide comprises a polynucleotide complementary to a targetsequence in the sample nucleic acid, and b) enzymatic activityconsisting of i) hyperthermophile polymerase activity and, optionally,ii) reverse transcriptase activity, thereby generating a nucleic acidamplification product.

Also provided herein in certain aspects are methods for processingnucleic acid, comprising amplifying nucleic acid, where the amplifyingconsists of contacting non-denatured sample nucleic acid underisothermal amplification conditions with a) at least oneoligonucleotide, which at least one oligonucleotide comprises apolynucleotide complementary to a target sequence in the sample nucleicacid, and b) at least one component providing hyperthermophilepolymerase activity, thereby generating a nucleic acid amplificationproduct.

Also provided herein in certain aspects are methods for processingnucleic acid, comprising amplifying nucleic acid, where the amplifyingconsists of contacting non-denatured sample nucleic acid underisothermal amplification conditions with a) non-enzymatic componentscomprising at least one oligonucleotide, which at least oneoligonucleotide comprises a polynucleotide complementary to a targetsequence in the sample nucleic acid, and b) an enzymatic componentconsisting of a hyperthermophile polymerase or a polymerase comprisingan amino acid sequence that is at least about 90% identical to ahyperthermophile polymerase, thereby generating a nucleic acidamplification product.

Also provided herein in certain aspects are methods for processingnucleic acid, comprising amplifying nucleic acid, where the amplifyingconsists of contacting non-denatured sample nucleic acid underisothermal amplification conditions with a) non-enzymatic componentscomprising at least one oligonucleotide, which at least oneoligonucleotide comprises a polynucleotide complementary to a targetsequence in the sample nucleic acid, and b) enzymatic activityconsisting of i) hyperthermophile polymerase activity and, optionally,ii) reverse transcriptase activity, thereby generating a nucleic acidamplification product.

Also provided herein in certain aspects are methods for determining thepresence, absence or amount of a target sequence in sample nucleic acid,comprising a) amplifying a target sequence in the sample nucleic acid,where the target sequence comprises a first strand and a second strand,the first strand and second strand are complementary to each other, andthe amplifying comprises contacting non-denatured sample nucleic acidunder helicase-free and/or recombinase-free isothermal amplificationconditions with i) a first oligonucleotide and a second oligonucleotide,where the first oligonucleotide comprises a first polynucleotidecontinuously complementary to a sequence in the first strand, and thesecond oligonucleotide comprises a second polynucleotide continuouslycomplementary to a sequence in the second strand; and ii) at least onecomponent providing a hyperthermophile polymerase activity, therebygenerating a nucleic acid amplification product, where the nucleic acidamplification product comprises 1) a first nucleotide sequence that iscontinuously complementary to or substantially identical to the firstpolynucleotide of the first oligonucleotide, 2) a second nucleotidesequence that is continuously complementary to or substantiallyidentical to the second polynucleotide of the second oligonucleotide,and 3) a spacer sequence comprising 1 to 10 bases, and the spacersequence is flanked by the first nucleotide sequence and the secondnucleotide sequence; and b) detecting the nucleic acid amplificationproduct, where detecting the nucleic acid amplification productcomprises use of a real-time detection method and is performed in 10minutes or less from the time the sample nucleic acid is contacted with(a)(i) and (a)(ii), whereby the presence, absence or amount of a targetsequence in sample nucleic acid is determined.

Also provided herein in certain aspects are methods for determining thepresence, absence or amount of a target sequence in sample nucleic acid,comprising a) amplifying a target sequence in the sample nucleic acid,which target sequence comprises a first strand and a second strand,which first strand and second strand are complementary to each other,where the amplifying comprises contacting non-denatured sample nucleicacid under helicase-free and/or recombinase-free isothermalamplification conditions with i) a first oligonucleotide and a secondoligonucleotide, where the first oligonucleotide consists of a firstpolynucleotide continuously complementary to a sequence in the firststrand, and the second oligonucleotide consists of a secondpolynucleotide continuously complementary to a sequence in the secondstrand; and ii) at least one component providing a hyperthermophilepolymerase activity, thereby generating a nucleic acid amplificationproduct, where the nucleic acid amplification product consists of 1) afirst nucleotide sequence that is continuously complementary to orsubstantially identical to the first polynucleotide of the firstoligonucleotide, 2) a second nucleotide sequence that is continuouslycomplementary to or substantially identical to the second polynucleotideof the second oligonucleotide, and 3) a spacer sequence comprising 1 to10 bases, where the spacer sequence is flanked by the first nucleotidesequence and the second nucleotide sequence; and b) detecting thenucleic acid amplification product, where detecting the nucleic acidamplification product comprises use of a real-time detection method andis performed in 10 minutes or less from the time the sample nucleic acidis contacted with (a)(i) and (a)(ii), whereby the presence, absence oramount of a target sequence in sample nucleic acid is determined.

Also provided herein in certain aspects are kits for determining thepresence, absence or amount of a target sequence in sample nucleic acidcomprising a) components for amplifying a target sequence in the samplenucleic acid under helicase-free and/or recombinase-free isothermalamplification conditions, which components comprise i) a firstoligonucleotide and a second oligonucleotide, where the firstoligonucleotide comprises a first polynucleotide continuouslycomplementary to a sequence in a first strand of the target sequence,and the second oligonucleotide comprises a second polynucleotidecontinuously complementary to a sequence in a second strand of thetarget sequence, which first strand and second strand of the targetsequence are complementary to each other; and ii) at least one componentproviding a hyperthermophile polymerase activity; and b) at least onecomponent providing real-time detection activity for a nucleic acidamplification product.

Also provided herein in certain aspects are kits for determining thepresence, absence or amount of a target sequence in sample nucleic acidcomprising a) components for amplifying a target sequence in the samplenucleic acid under helicase-free and/or recombinase-free isothermalamplification conditions, which components comprise i) a firstoligonucleotide and a second oligonucleotide, where the firstoligonucleotide consists of a first polynucleotide continuouslycomplementary to a sequence in a first strand of the target sequence,and the second oligonucleotide consists of a second polynucleotidecontinuously complementary to a sequence in a second strand of thetarget sequence, which first strand and second strand of the targetsequence are complementary to each other; and ii) at least one componentproviding a hyperthermophile polymerase activity; and b) at least onecomponent providing real-time detection activity for a nucleic acidamplification product.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate certain embodiments of the technology and arenot limiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 shows real-time detection of chlamydia genomic DNA amplificationreactions performed in duplicate with dH₂O or Tris-EDTA buffer (TE) usedas a no target control (NTC; negative control).

FIG. 2 shows Electrospray Ionization Mass Spectrometry (ESI-MS)detection of chlamydia genomic DNA assay products.

FIG. 3 shows chlamydia genomic DNA limit of detection (LOD) by endpointmolecular beacon detection.

FIG. 4 shows chlamydia genomic DNA real-time detection by molecularbeacon.

FIG. 5 shows a schematic of an isothermal amplification reactiondescribed herein.

DETAILED DESCRIPTION

Provided herein are methods and compositions for amplifying nucleicacid. Traditional nucleic acid amplification methods typically require athermocycling process, nucleic acid denaturation, proteins (e.g.,enzymes) that promote strand unwinding, strand separation and/or strandexchange (e.g., helicases, recombinases), and/or endonuclease agents(e.g., restriction enzymes, nicking enzymes), and often require aminimum reaction time of 20 to 30 minutes. The nucleic acidamplification methods provided herein can be performed withoutthermocycling, without nucleic acid denaturation, without added proteins(e.g., enzymes) to promote strand unwinding, strand separation and/orstrand exchange, without endonuclease agents, and within a reaction timeof 10 minutes.

Nucleic Acid, Subjects, Samples and Nucleic Acid Processing

Provided herein are methods and compositions for amplifying nucleicacid. The terms “nucleic acid” and “nucleic acid molecule” may be usedinterchangeably herein. The terms refer to nucleic acids of anycomposition, such as DNA (e.g., complementary DNA (cDNA), genomic DNA(gDNA) and the like), RNA (e.g., message RNA (mRNA), short inhibitoryRNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and/or DNA or RNAanalogs (e.g., containing base analogs, sugar analogs and/or anon-native backbone and the like), RNA/DNA hybrids and polyamide nucleicacids (PNAs), all of which can be in single- or double-stranded form,and unless otherwise limited, can encompass known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides. A nucleic acid may be, or may be from, a plasmid, phage,autonomously replicating sequence (ARS), centromere, artificialchromosome, chromosome, or other nucleic acid able to replicate or bereplicated in vitro or in a host cell, a cell, a cell nucleus, amitochondria, or cytoplasm of a cell in certain embodiments. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogs of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms(SNPs), and complementary sequences as well as the sequence explicitlyindicated. Specifically, degenerate codon substitutions may be achievedby generating sequences in which the third position of one or moreselected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues. The term nucleic acid may be used interchangeablywith locus, gene, cDNA, and mRNA encoded by a gene. The term also mayinclude, as equivalents, derivatives, variants and analogs of RNA or DNAsynthesized from nucleotide analogs, single-stranded (“sense” or“antisense”, “plus” strand or “minus” strand, “forward” reading frame or“reverse” reading frame, “forward” strand or “reverse” strand) anddouble-stranded polynucleotides. The term “gene” means the segment ofDNA involved in producing a polypeptide chain; and generally includesregions preceding and following the coding region (leader and trailer)involved in the transcription/translation of the gene product and theregulation of the transcription/translation, as well as interveningsequences (introns) between individual coding segments (exons). Anucleotide or base generally refers to the purine and pyrimidinemolecular units of nucleic acid (e.g., adenine (A), thymine (T), guanine(G), and cytosine (C)). For RNA, the base thymine is replaced withuracil. Nucleic acid length or size may be expressed as a number ofbases.

In some embodiments of the methods provided herein, one or more nucleicacid targets are amplified. Target nucleic acids may be referred to astarget sequences, target polynucleotides, and/or target polynucleotidesequences, and may include double-stranded and single-stranded nucleicacid molecules. Target nucleic acid may be, for example, DNA or RNA.Where the target nucleic acid is an RNA molecule, the molecule may be,for example, double-stranded, single-stranded, or the RNA molecule maycomprise a target sequence that is single-stranded. Where the targetnucleic acid is double stranded, the target nucleic acid generallyincludes a first strand and a second strand. A first strand and a secondstrand may be referred to as a forward strand and a reverse strand andgenerally are complementary to each other. Where the target nucleic acidis single stranded, a complementary strand may be generated, for exampleby polymerization and/or reverse transcription, rendering the targetnucleic acid double stranded and having a first/forward strand and asecond/reverse strand.

A target sequence may refer to either the sense or antisense strand of anucleic acid sequence, and also may refer to sequences as they exist ontarget nucleic acids, amplified copies, or amplification products, ofthe original target sequence. A target sequence may be a subsequencewithin a larger polynucleotide. For example, a target sequence may be ashort sequence (e.g., 20 to 50 bases) within a nucleic acid fragment, achromosome, a plasmid, that is targeted for amplification. In someembodiments, a target sequence may refer to a sequence in a targetnucleic acid that is complementary to an oligonucleotide (e.g., primer)used for amplifying a nucleic acid. Thus, a target sequence may refer tothe entire sequence targeted for amplification or may refer to asubsequence in the target nucleic acid where an oligonucleotide binds.An amplification product may be a larger molecule that comprises thetarget sequence, as well as at least one other sequence, or othernucleotides. In some embodiments, an amplification product is about thesame length as the target sequence. In some embodiments, anamplification product is exactly the same length as the target sequence.In some embodiments, an amplification product comprises the targetsequence. In some embodiments, an amplification product consists of thetarget sequence.

The length of the target sequence, and/or the guanosine cytosine (GC)concentration (percent), may depend, in part, on the temperature atwhich an amplification reaction is run, and this temperature may depend,in part, on the stability of the polymerase(s) used in the reaction.Sample assays may be performed to determine an appropriate targetsequence length and GC concentration for a set of reaction conditions.For example, where a polymerase is stable up to 60° C. to 65° C., thenthe target sequence may be, for example, from 19 to 50 nucleotides inlength, or for example, from about 40 to 50, 20 to 45, 20 to 40, or 20to 30 nucleotides in length. GC concentration under these conditions maybe, for example, less than 60%, less than 55%, less than 50%, or lessthan 45%.

Target nucleic acid may include, for example, genomic nucleic acid,plasmid nucleic acid, mitochondrial nucleic acid, cellular nucleic acid,extracellular nucleic acid, bacterial nucleic acid and viral nucleicacid. In some embodiments, target nucleic acid may include genomic DNA,chromosomal DNA, plasmid DNA, mitochondrial DNA, a gene, any type ofcellular RNA, messenger RNA, bacterial RNA, viral RNA or a syntheticoligonucleotide. Genomic nucleic acid may include any nucleic acid fromany genome, for example, including animal, plant, insect, viral andbacterial genomes, including, for example, genomes present in spores. Insome embodiments, genomic target nucleic acid may be within a particulargenomic locus or a plurality of genomic loci. A genomic locus mayinclude any or a combination of open reading frame DNA, non-transcribedDNA, intronic sequences, extronic sequences, promoter sequences,enhancer sequences, flanking sequences, or any sequences consideredassociated with a given genomic locus.

In some embodiments, a target sequence comprises one or more repetitiveelements (e.g., multiple repeat sequences, inverted repeat sequences,palindromic sequences, tandem repeats, microsatellites, minisatellites,and the like). In some embodiments, a target sequence is present withina sample nucleic acid (e.g., within a nucleic acid fragment, achromosome, a genome, a plasmid) as a repetitive element (e.g., amultiple repeat sequence, an inverted repeat sequence, a palindromicsequence, a tandem repeat, a microsatellite repeat, a minisatelliterepeat and the like). For example, a target sequence may occur multipletimes as a repetitive element and one, some, or all occurrences of thetarget sequence within a repetitive element may be amplified (e.g.,using a single pair of primers) using methods described herein. In someembodiments, a target sequence is present within a sample nucleic acid(e.g., within a nucleic acid fragment, a chromosome, a genome, aplasmid) as a duplication and/or a paralog.

Target nucleic acid may include microRNAs. MicroRNAs, miRNAs, or smalltemporal RNAs (stRNAs) are short (e.g., about 21 to 23 nucleotides long)and single-stranded RNA sequences involved in gene regulation. MicroRNAsmay interfere with translation of messenger RNAs and are partiallycomplementary to messenger RNAs. Target nucleic acid may includemicroRNA precursors such as primary transcript (pri-miRNA) and pre-miRNAstem-loop-structured RNA that is further processed into miRNA. Targetnucleic acid may include short interfering RNAs (siRNAs), which areshort (e.g., about 20 to 25 nucleotides long) and at least partiallydouble-stranded RNA molecules involved in RNA interference (e.g.,down-regulation of viral replication or gene expression).

Nucleic acid utilized in methods described herein may be obtained fromany suitable biological specimen or sample, and often is isolated from asample obtained from a subject. A subject can be any living ornon-living organism, including but not limited to a human, a non-humananimal, a plant, a bacterium, a fungus, a virus, or a protist. Any humanor non-human animal can be selected, including but not limited tomammal, reptile, avian, amphibian, fish, ungulate, ruminant, bovine(e.g., cattle), equine (e.g., horse), caprine and ovine (e.g., sheep,goat), swine (e.g., pig), camelid (e.g., camel, llama, alpaca), monkey,ape (e.g., gorilla, chimpanzee), ursid (e.g., bear), poultry, dog, cat,mouse, rat, fish, dolphin, whale and shark. A subject may be a male orfemale, and a subject may be any age (e.g., an embryo, a fetus, infant,child, adult).

A sample or test sample can be any specimen that is isolated or obtainedfrom a subject or part thereof. Non-limiting examples of specimensinclude fluid or tissue from a subject, including, without limitation,blood or a blood product (e.g., serum, plasma, or the like), umbilicalcord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinalfluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric,peritoneal, ductal, ear, arthroscopic), biopsy sample, celocentesissample, cells (e.g., blood cells) or parts thereof (e.g., mitochondrial,nucleus, extracts, or the like), washings of female reproductive tract,urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage,semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid,hard tissues (e.g., liver, spleen, kidney, lung, or ovary), the like orcombinations thereof. The term blood encompasses whole blood, bloodproduct or any fraction of blood, such as serum, plasma, buffy coat, orthe like as conventionally defined. Blood plasma refers to the fractionof whole blood resulting from centrifugation of blood treated withanticoagulants. Blood serum refers to the watery portion of fluidremaining after a blood sample has coagulated. Fluid or tissue samplesoften are collected in accordance with standard protocols hospitals orclinics generally follow. For blood, an appropriate amount of peripheralblood (e.g., between 3-40 milliliters) often is collected and can bestored according to standard procedures prior to or after preparation.

A sample or test sample can include samples containing spores, viruses,cells, nucleic acid from prokaryotes or eukaryotes, or any free nucleicacid. For example, a method described herein may be used for detectingnucleic acid on the outside of spores (e.g., without the need forlysis). A sample may be isolated from any material suspected ofcontaining a target sequence, such as from a subject described above. Incertain instances, a target sequence may be present in air, plant, soil,or other materials suspected of containing biological organisms.

Nucleic acid may be derived (e.g., isolated, extracted, purified) fromone or more sources by methods known in the art. Any suitable method canbe used for isolating, extracting and/or purifying nucleic acid from abiological sample, non-limiting examples of which include methods of DNApreparation in the art, and various commercially available reagents orkits, such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNAMini Kit or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany),GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Ws.), GFX™Genomic Blood DNA Purification Kit (Amersham, Piscataway, N.J.), and thelike or combinations thereof.

In some embodiments, a cell lysis procedure is performed. Cell lysis maybe performed prior to initiation of an amplification reaction describedherein (e.g., to release DNA and/or RNA from cells for amplification).Cell lysis procedures and reagents are known in the art and maygenerally be performed by chemical (e.g., detergent, hypotonicsolutions, enzymatic procedures, and the like, or combination thereof),physical (e.g., French press, sonication, and the like), or electrolyticlysis methods. Any suitable lysis procedure can be utilized. Forexample, chemical methods generally employ lysing agents to disruptcells and extract nucleic acids from the cells, followed by treatmentwith chaotropic salts. In some embodiments, cell lysis comprises use ofdetergents (e.g., ionic, nonionic, anionic, zwitterionic). In someembodiments, cell lysis comprises use of ionic detergents (e.g., sodiumdodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate,cholate, sarkosyl) Physical methods such as freeze/thaw followed bygrinding, the use of cell presses and the like also may be useful. Highsalt lysis procedures also may be used. For example, an alkaline lysisprocedure may be utilized. The latter procedure traditionallyincorporates the use of phenol-chloroform solutions, and an alternativephenol-chloroform-free procedure involving three solutions may beutilized. In the latter procedures, one solution can contain 15 mM Tris,pH 8.0; 10 mM EDTA and 100 μg/ml Rnase A; a second solution can contain0.2N NaOH and 1% SDS; and a third solution can contain 3M KOAc, pH 5.5,for example. In some embodiments, a cell lysis buffer is used inconjunction with the methods and components described herein.

Nucleic acid may be provided for conducting methods described hereinwithout processing of the sample(s) containing the nucleic acid, incertain embodiments. For example, in some embodiments, nucleic acid isprovided for conducting amplification methods described herein withoutprior nucleic acid purification. In some embodiments, a target sequenceis amplified directly from a sample (e.g., without performing anynucleic acid extraction, isolation, purification and/or partialpurification steps). In some embodiments, nucleic acid is provided forconducting methods described herein after processing of the sample(s)containing the nucleic acid. For example, a nucleic acid can beextracted, isolated, purified, or partially purified from the sample(s).The term “isolated” generally refers to nucleic acid removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring, or a host cell if expressed exogenously), and thus is alteredby human intervention (e.g., “by the hand of man”) from its originalenvironment. The term “isolated nucleic acid” can refer to a nucleicacid removed from a subject (e.g., a human subject). An isolated nucleicacid can be provided with fewer non-nucleic acid components (e.g.,protein, lipid, carbohydrate) than the amount of components present in asource sample. A composition comprising isolated nucleic acid can beabout 50% to greater than 99% free of non-nucleic acid components. Acomposition comprising isolated nucleic acid can be about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free ofnon-nucleic acid components. The term “purified” generally refers to anucleic acid provided that contains fewer non-nucleic acid components(e.g., protein, lipid, carbohydrate) than the amount of non-nucleic acidcomponents present prior to subjecting the nucleic acid to apurification procedure. A composition comprising purified nucleic acidmay be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of othernon-nucleic acid components.

Nucleic acid may be provided for conducting methods described hereinwithout modifying the nucleic acid. Modifications may include, forexample, denaturation, digestion, nicking, unwinding, incorporationand/or ligation of heterogeneous sequences, addition of epigeneticmodifications, addition of labels (e.g., radiolabels such as ³²P, ³³P,¹²⁵I, or ³⁵S; enzyme labels such as alkaline phosphatase; fluorescentlabels such as fluorescein isothiocyanate (FITC); or other labels suchas biotin, avidin, digoxigenin, antigens, haptens, fluorochromes), andthe like. Accordingly, in some embodiments, an unmodified nucleic acidis amplified.

Amplification

Provided herein are methods for amplifying nucleic acid. In someembodiments, nucleic acids are amplified using a suitable amplificationprocess. Nucleic acid amplification typically involves enzymaticsynthesis of nucleic acid amplicons (copies), which contain a sequencecomplementary to a nucleotide sequence being amplified. In someembodiments, an amplification method is performed in a single vessel, asingle chamber, and/or a single volume (i.e., contiguous volume). Insome embodiments, an amplification method and a detection method (e.g.,such as a detection method described herein) are performed in a singlevessel, a single chamber, and/or a single volume (i.e., contiguousvolume).

The terms “amplify”, “amplification”, “amplification reaction”, or“amplifying” refer to any in vitro process for multiplying the copies ofa target nucleic acid. Amplification sometimes refers to an“exponential” increase in target nucleic acid. However, “amplifying” mayalso refer to linear increases in the numbers of a target nucleic acid,but is different than a one-time, single primer extension step. In someembodiments a limited amplification reaction, also known aspre-amplification, can be performed. Pre-amplification is a method inwhich a limited amount of amplification occurs due to a small number ofcycles, for example 10 cycles, being performed. Pre-amplification canallow some amplification, but stops amplification prior to theexponential phase, and typically produces about 500 copies of thedesired nucleotide sequence(s). Use of pre-amplification may limitinaccuracies associated with depleted reactants in certain amplificationreactions, and also may reduce amplification biases due to nucleotidesequence or species abundance of the target. In some embodiments aone-time primer extension may be performed as a prelude to linear orexponential amplification.

A generalized description of an amplification process is presentedherein. Primers (e.g., oligonucleotides described herein) and targetnucleic acid are contacted, and complementary sequences anneal orhybridize to one another, for example. Primers can anneal to a targetnucleic acid, at or near (e.g., adjacent to, abutting, and the like) asequence of interest. A primer annealed to a target may be referred toas a primer-target hybrid, hybridized primer-target, or a primer-targetduplex. The terms near or adjacent to when referring to a nucleotidesequence of interest refer to a distance (e.g., number of bases) orregion between the end of the primer and the nucleotide or nucleotides(e.g., nucleotide sequence) of a target. Generally, adjacent is in therange of about 1 nucleotide to about 50 nucleotides (e.g., 1 nucleotide,2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, about 10nucleotides, about 20 nucleotides, about 30 nucleotides, about 40nucleotides, about 50 nucleotides) away from a nucleotide or nucleotidesequence of interest. In some embodiments, primers in a set (e.g., apair of primers, a forward and a reverse primer, a first oligonucleotideand a second oligonucleotide) anneal within about 1 to 20 nucleotidesfrom a nucleotide or nucleotide sequence of interest and produceamplified products. In some embodiments, primers anneal within anucleotide or a nucleotide sequence of interest. After annealing, eachprimer is extended along the target (i.e., template strand) by apolymerase to generate a complementary strand. Several cycles of primerannealing and extension may be carried out, for example, until adetectable amount of amplification product is generated. In someembodiments, where a target nucleic acid is RNA, a DNA copy (cDNA) ofthe target RNA may be synthesized prior to or during the amplificationstep by reverse transcription.

Components of an amplification reaction may include, for example, one ormore primers (e.g., individual primers, primer pairs, primer sets,oligonucleotides, multiple primer sets for multiplex amplification, andthe like), nucleic acid target(s) (e.g., target nucleic acid from asample), one or more polymerases, nucleotides (e.g., dNTPs and thelike), and a suitable buffer (e.g., a buffer comprising a detergent, areducing agent, monovalent ions, and divalent ions). An amplificationreaction may further include a reverse transcriptase, in someembodiments. An amplification reaction may further include one or moredetection agents, such as one or more of the detection agents describedherein, in some embodiments. In some embodiments, components of anamplification reaction consist of primers, target nucleic acid, apolymerase, nucleotides, and a suitable buffer. In some embodiments,components of an amplification reaction consist of primers, targetnucleic acid, a polymerase, a reverse transcriptase, nucleotides, and asuitable buffer. In some embodiments, components of an amplificationreaction consist of primers, target nucleic acid, a polymerase, adetection agent, nucleotides, and a suitable buffer. In someembodiments, components of an amplification reaction consist of primers,target nucleic acid, a polymerase, a reverse transcriptase, a detectionagent, nucleotides, and a suitable buffer. In some embodiments,components of an amplification reaction consist essentially of primers,target nucleic acid, a polymerase, nucleotides, and a suitable buffer.In some embodiments, components of an amplification reaction consistessentially of primers, target nucleic acid, a polymerase, a reversetranscriptase, nucleotides, and a suitable buffer. In some embodiments,components of an amplification reaction consist essentially of primers,target nucleic acid, a polymerase, a detection agent, nucleotides, and asuitable buffer. In some embodiments, components of an amplificationreaction consist essentially of primers, target nucleic acid, apolymerase, a reverse transcriptase, a detection agent, nucleotides, anda suitable buffer. When components of an amplification reaction consistessentially of certain components, additional components or features maybe included that do not have a significant effect on the amplificationand/or are not necessary for generating a detectable product. Forexample, additional components or features may be included that do nothave a significant effect on the ability of the components andconditions herein to achieve amplification under isothermal conditionsand generate a detectable amplification product within about 10 minutesor less. Such additional components or features may be referred to asnon-essential components and may include typical reaction componentsand/or common additives such as salts, buffers, detergents, ions, oils,proteins, polymers and the like.

Nucleic acid amplification may be conducted in the presence of nativenucleotides, such as, for example, dideoxyribonucleoside triphosphates(dNTPs), and/or derivatized nucleotides. A native nucleotide generallyrefers to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid,or uridylic acid. A derivatized nucleotide generally is a nucleotideother than a native nucleotide. Nucleotides typically are designated asfollows. A ribonucleoside triphosphate is referred to as NTP or rNTP,where N can be A, G, C, U. A deoxynucleoside triphosphate substrates isreferred to as dNTP, where N can be A, G, C, T, or U. Monomericnucleotide subunits may be denoted as A, G, C, T, or U herein with noparticular reference to DNA or RNA. In some embodiments, non-naturallyoccurring nucleotides or nucleotide analogs, such as analogs containinga detectable label (e.g., fluorescent or colorimetric label), may beused. For example, nucleic acid amplification may be carried out in thepresence of labeled dNTPs, such as, for example, radiolabels such as³²P, ³³P, ¹²⁵I, or ³⁵S; enzyme labels such as alkaline phosphatase;fluorescent labels such as fluorescein isothiocyanate (FITC); or otherlabels such as biotin, avidin, digoxigenin, antigens, haptens, orfluorochromes. In some embodiments, nucleic acid amplification may becarried out in the presence of modified dNTPs, such as, for example,heat activated dNTPs (e.g., CleanAmp™ dNTPs from Tri Link).

In some embodiments, components of an amplification reaction may includenon-enzymatic components and enzymatic components. Non-enzymaticcomponents may include, for example, primers, nucleotides, buffers,salts, reducing agents, detergents, and ions; and generally do notinclude proteins (e.g., nucleic acid binding proteins), enzymes, orproteins having enzymatic activity such as, for example, polymerases,reverse transcriptases, helicases, topoisomerases, ligases,exonucleases, endonucleases, restriction enzymes, nicking enzymes,recombinases and the like. In some embodiments, an enzymatic componentmay consist of a polymerase or may consist of a polymerase and a reversetranscriptase. Accordingly, such enzymatic components would excludeother proteins (e.g., nucleic acid binding proteins and/or proteinshaving enzymatic activity) such as, for example, helicases,topoisomerases, ligases, exonucleases, endonucleases, restrictionenzymes, nicking enzymes, recombinases, and the like.

In some embodiments, amplification conditions comprise an enzymaticactivity. Typically, an enzymatic activity is provided by a polymerase,and in certain instances, an enzymatic activity is provided by apolymerase and a reverse transcriptase. In some embodiments, anenzymatic activity consists of a polymerase activity. In someembodiments, an enzymatic activity consists of a polymerase activity anda reverse transcriptase activity. Accordingly, in some embodiments,enzymatic activity does not include enzymatic activity provided by otherenzymes such as, for example, helicases, topoisomerases, ligases,exonucleases, endonucleases, restriction enzymes, nicking enzymes,recombinases, and the like. In certain instances, a polymerase activityand a reverse transcriptase activity are provided by separate enzymes orseparate enzyme types (e.g., polymerase(s) and reversetranscriptase(s)). In certain instances, a polymerase activity and areverse transcriptase activity are provided by a single enzyme or enzymetype (e.g., polymerase(s)).

In some embodiments, amplification of nucleic acid comprises anon-thermocycling type of polymerase chain reaction (PCR). In someembodiments, amplification of nucleic acid comprises an isothermalamplification process. In some embodiments, amplification of nucleicacid comprises an isothermal polymerase chain reaction (iPCR).Isothermal amplification generally is an amplification process performedat a constant temperature. Terms such as isothermal conditions,isothermally and constant temperature generally refer to reactionconditions where the temperature of the reaction is kept essentiallyconstant during the course of the amplification reaction. Isothermalamplification conditions generally do not include a thermocycling (i.e.,cycling between an upper temperature and a lower temperature) componentin the amplification process. When amplifying under isothermalconditions, the reaction may be kept at an essentially constanttemperature, which means the temperature may not be maintained atprecisely one temperature. For example, small fluctuations intemperature (e.g., ±1 to 5 degrees Celsius) may occur in an isothermalamplification process due to, for example, environmental orequipment-based variables. Often, the entire reaction volume is kept atan essentially constant temperature, and isothermal reactions hereingenerally do not include amplification conditions that rely on atemperature gradient generated within a reaction vessel and/orconvective-flow based temperature cycling.

Isothermal amplification reactions herein may be conducted at anessentially constant temperature. In some embodiments, isothermalamplification reactions herein are conducted at a temperature of about55 degrees Celsius to a temperature of about 75 degrees Celsius. Forexample, isothermal amplification reactions herein may be conducted at atemperature of about 55 degrees Celsius, about 56 degrees Celsius, about57 degrees Celsius, about 58 degrees Celsius, about 59 degrees Celsius,about 60 degrees Celsius, about 61 degrees Celsius, about 62 degreesCelsius, about 63 degrees Celsius, about 64 degrees Celsius, about 65degrees Celsius, about 66 degrees Celsius, about 67 degrees Celsius,about 68 degrees Celsius, about 69 degrees Celsius, about 70 degreesCelsius, about 71 degrees Celsius, about 72 degrees Celsius, about 73degrees Celsius, about 74 degrees Celsius, or about 75 degrees Celsius.In some embodiments, isothermal amplification reactions herein areconducted at a temperature of about 55 degrees Celsius to a temperatureof about 65 degrees Celsius. For example, isothermal amplificationreactions herein may be conducted at a temperature of about 60 degreesCelsius. Isothermal amplification reactions herein may be conducted at atemperature of about 65 degrees Celsius. In some embodiments, atemperature element (e.g., heat source) is kept at an essentiallyconstant temperature. In some embodiments, a temperature element is keptat an essentially constant temperature at or below about 75 degreesCelsius. In some embodiments, a temperature element is kept at anessentially constant temperature at or below about 70 degrees Celsius.In some embodiments, a temperature element is kept at an essentiallyconstant temperature at or below about 65 degrees Celsius. In someembodiments, a temperature element is kept at an essentially constanttemperature at or below about 60 degrees Celsius.

An amplification process herein may be conducted over a certain lengthof time. In some embodiments, an amplification process is conducteduntil a detectable nucleic acid amplification product is generated. Anucleic acid amplification product may be detected by any suitabledetection process and/or a detection process described herein. In someembodiments, an amplification process is conducted over a length of timewithin about 20 minutes or less. For example, an amplification processmay be conducted within about 1 minute, about 2 minutes, about 3minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19minutes, or about 20 minutes. In some embodiments, an amplificationprocess is conducted over a length of time within about 10 minutes orless.

Nucleic acid targets may be amplified without exposure to agents orconditions that denature nucleic acid, in some embodiments. Agents orconditions that denature nucleic acid may include agents or conditionsthat promote strand separation and/or promote unwinding. Nucleic acidtargets may be amplified without exposure to agents or conditions thatpromote strand separation, in some embodiments. Nucleic acid targets maybe amplified without exposure to agents or conditions that promoteunwinding, in some embodiments. In some embodiments, a target nucleicacid is considered non-denatured if it has not been exposed to agents orconditions that denature nucleic acid and/or promote strand separationand/or promote unwinding prior to or during amplification. Agents orconditions that denature nucleic acid and/or promote strand separationand/or promote unwinding may include, for example, thermal conditions(e.g., high temperatures), pH conditions (e.g., high or low pH),chemical agents, proteins (e.g., enzymatic agents), and the like.

Nucleic acid targets may be amplified without exposure to agents orconditions that denature nucleic acid, in some embodiments. Nucleic aciddenaturation, or melting, is the process by which double-strandednucleic acid unwinds and separates into single strands. Agents andconditions that can promote nucleic acid denaturation include, forexample, heat, high pH, low pH, and denaturing agents (e.g., formamide)combined with heat. In some instances, denaturation can be achieved byheating a solution containing nucleic acid to a certain temperature, forexample a temperature above 75 degrees Celsius, above 80 degreesCelsius, above 90 degrees Celsius, above 95 degrees Celsius, or higher.In some instances, denaturation can be achieved by exposure todenaturing agents, such as, for example NaOH, HCl, and formamidecombined with heat. Specific methods for DNA denaturation are described,for example, in Singh et al., (1977) Chomosoma 60:377-389.

Nucleic acid targets may be amplified without exposure to agents orconditions that promote strand separation and/or unwinding, in someembodiments. For example, nucleic acid targets may be amplified withoutexposure to a helicase. Helicases are enzymes capable of unwinding andseparating double-stranded nucleic acid into single strands. Examples ofhelicases include human DNA helicases (and their equivalents in otherorganisms) such as DNA helicase Q1, Bloom syndrome protein, Wernersyndrome protein, DNA helicase Q4, DNA helicase Q5, DNA helicase 2subunit 1, MCM2, MCM3, MCM, MCMS, MCM6, MCM7, MCM8, MCM9, MCM10,Nucleolin, CHD2, CHD7, XPB, XPD, lymphoid-specific helicase, hINO,RuvB-like 1, RuvB-like 2, PIF1, Twinkle, BACH1, RecQ5 alpha, RecQ5 beta,RecQ5 gamma and RTEL1; human RNA helicases (and their equivalents inother organisms) such as RNA helicase DDX1, RNA helicase eIF4A-1, RNAhelicase eIF4A-2, RNA helicase DDX3X, RNA helicase DDX3Y, RNA helicaseDDX4, RNA helicase DDX5, RNA helicase DDX6, RNA helicase DHX8, RNAhelicase A, RNA helicase DDX10, RNA helicase DDX11, RNA helicase DDX12,Helicase SKI2W, RNA helicase DHX15, RNA helicase DHX16, RNA helicaseDDX17, RNA helicase DDX18, RNA helicase DDX19A, RNA helicase DDX19B, RNAhelicase DDX20, Nucleolar RNA helicase 2, RNA helicase DDX23, RNAhelicase DDX24, RNA helicase DDX25, RNA helicase DDX27, RNA helicaseDDX28, RNA helicase DHX29, RNA helicase DHX30, RNA helicase DDX31, RNAhelicase DHX32, RNA helicase DHX33, RNA helicase DHX34, RNA helicaseDHX35, RNA helicase DHX36, RNA helicase DHX37, RNA helicase PRP 16, RNAhelicase DDX39, RNA helicase DHX40, RNA helicase DDX41, RNA helicaseDDX42, RNA helicase DDX43, RNA helicase DDX46, RNA helicase DDX47, RNAhelicase eIF4A-3, RNA helicase DDX49, RNA helicase DDX50, RNA helicaseDDX51, RNA helicase DDX52, RNA helicase DDX53, RNA helicase DDX54, RNAhelicase DDX55, RNA helicase DDX56, RNA helicase DHX57, RNA helicaseDDX58, RNA helicase DHX58, RNA helicase DDX59, RNA helicase DDX60,Spliceosome RNA helicase BAT1, U5.snRNP 200 kDa helicase,Transcriptional regulator ATRX helicase, RNA helicase SUPV3L1,mitochondrial Superkiller viralicidic activity 2-like 2, and Fanconianemia group J protein; and commercially available helicases.Amplification conditions that do not include use of a helicase may bereferred to herein as helicase-free amplification conditions.

In some embodiments, nucleic acid targets may be amplified withoutexposure to a recombinase. Recombinases are enzymes involved in geneticrecombination and sometimes are involved in nucleic acid repair (e.g.,recombinational DNA repair). Recombinases can initiate strand exchange,for example. Recombinases may include, for example, Cre recombinase, Hinrecombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, T4uvsX. In some embodiments, nucleic acid targets may be amplified withoutexposure to a recombinase accessory protein, such as, for example, arecombinase loading factor (e.g., T4 uvsY).

In some embodiments, nucleic acid targets may be amplified withoutexposure to a nucleic acid binding protein (e.g., single-strandedbinding protein or single-strand DNA-binding protein (SSB)). Singlestranded binding proteins generally function to prevent prematureannealing, protect the single-stranded DNA from being digested bynucleases, and/or remove secondary structure from DNA (e.g., destabilizehelical duplexes) to allow enable action by other enzymes. In someembodiments, nucleic acid targets may be amplified without exposure to asingle-strand DNA-binding protein, such as, for example, T4 gp32.

In some embodiments, nucleic acid targets may be amplified withoutexposure to a topoisomerase. Topoisomerases are enzymes that regulatethe overwinding or underwinding of DNA by binding to eithersingle-stranded or double-stranded DNA and cutting the DNA phosphatebackbone. Amplification conditions that do not include use of atopoisomerase may be referred to herein as topoisomerase-freeamplification conditions.

Nucleic acid targets may be amplified with or without exposure to agentsor conditions that destabilize nucleic acid. Destabilization generallyrefers to a disruption in the overall organization and geometricorientation of a nucleic acid molecule (e.g., double helical structure)by one or more of tilt, roll, twist, slip, and flip effects (e.g., asdescribed in Lenglet et al., (2010) Journal of Nucleic Acids Volume2010, Article ID 290935, 17 pages). Destabilization generally does notrefer to melting or separation of nucleic acid strands, as describedabove for denaturation. Nucleic acid destabilization may be achieved,for example, by exposure to agents such as intercalators or alkylatingagents, and/or chemicals such as formamide, urea, dimethyl sulfoxide(DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments,amplification methods may include use of one or more destabilizingagents. In some embodiments, amplification methods exclude use ofdestabilizing agents.

In some embodiments, nucleic acid targets may be amplified withoutexposure to a ligase. Ligases are enzyme that can catalyze the joiningof amino acid molecules by forming a new chemical bond. Amplificationconditions that do not include use of a ligase may be referred to hereinas ligase-free amplification conditions.

In some embodiments, nucleic acid targets may be amplified withoutexposure to an RNA replicase. RNA replicases, RNA-dependent RNApolymerase (RdRp), or RDR, are enzymes that catalyze the replication ofRNA from an RNA template. Amplification conditions that do not includeuse of an RNA replicase may be referred to herein as RNA replicase-freeamplification conditions.

Nucleic acid targets may be amplified without cleavage or digestion, incertain embodiments. For example, in some embodiments, nucleic acid isamplified without prior exposure to one or more cleavage agents, andintact nucleic acid is amplified. In certain embodiments, nucleic acidis amplified without exposure to one or more cleavage agents duringamplification. In certain embodiments, nucleic acid is amplified withoutexposure to one or more cleavage agents after amplification.Amplification conditions that do not include use of a cleavage agent maybe referred to herein as cleavage agent-free amplification conditions.The term “cleavage agent” generally refers to an agent, sometimes achemical or an enzyme that can cleave a nucleic acid at one or morespecific or non-specific sites. Specific cleavage agents often cleavespecifically according to a particular nucleotide sequence at aparticular site. Cleavage agents may include endonucleases (e.g.,restriction enzymes, nicking enzymes, and the like); exonucleases(DNAses, RNAses (e.g., RNAseH), 5′ to 3′ exonucleases (e.g. exonucleaseII), 3′ to 5′ exonucleases (e.g. exonuclease I), and poly(A)-specific 3′to 5′ exonucleases); and chemical cleaving agents.

Nucleic acid targets may be amplified without use of restriction enzymesand/or nicking enzymes, in certain embodiments. A restriction enzyme isa protein that cuts DNA at a specific site and generally cleaves bothstrands of a double-stranded duplex, and a nicking enzyme is a proteinthat binds to double-stranded DNA and cleaves one strand of adouble-stranded duplex. In certain embodiments, nucleic acid isamplified without prior exposure to restriction enzymes and/or nickingenzymes. In certain embodiments, nucleic acid is amplified withoutexposure to restriction enzymes and/or nicking enzymes duringamplification. In certain embodiments, nucleic acid is amplified withoutexposure to restriction enzymes and/or nicking enzymes afteramplification. Amplification conditions that do not include use of arestriction enzyme may be referred to herein as restriction enzyme-freeamplification conditions. Amplification conditions that do not includeuse of a nicking enzyme may be referred to herein as nicking enzyme-freeamplification conditions.

Nucleic acid targets may be amplified without exonuclease treatment, incertain embodiments. Exonucleases are enzymes that work by cleavingnucleotides one at a time from the end of a polynucleotide chain througha hydrolyzing reaction that breaks phosphodiester bonds at either the 3′or the 5′ end. Exonucleases include, for example, DNAses, RNAses (e.g.,RNAseH), 5′ to 3′ exonucleases (e.g. exonuclease II), 3′ to 5′exonucleases (e.g. exonuclease I), and poly(A)-specific 3′ to 5′exonucleases. In certain embodiments, nucleic acid is amplified withoutexonuclease treatment prior to amplification. In certain embodiments,nucleic acid is amplified without exonuclease treatment duringamplification. In certain embodiments, nucleic acid is amplified withoutexonuclease after amplification. Amplification conditions that do notinclude use of an exonuclease may be referred to herein asexonuclease-free amplification conditions. In certain embodiments,nucleic acid is amplified without DNAse treatment. In certainembodiments, nucleic acid is amplified without RNAse treatment. Incertain embodiments, nucleic acid is amplified without RNAseH treatment.Amplification conditions that do not include use of DNAse may bereferred to herein as DNAse-free amplification conditions. Amplificationconditions that do not include use of RNAse may be referred to herein asRNAse-free amplification conditions. Amplification conditions that donot include use of RNAseH may be referred to herein as RNAseH-freeamplification conditions.

An amplified nucleic acid may be referred to herein as a nucleic acidamplification product or amplicon. In some embodiments, an amplificationproduct may include naturally occurring nucleotides, non-naturallyoccurring nucleotides, nucleotide analogs and the like and combinationsof the foregoing. An amplification product typically has a nucleotidesequence that is identical to or substantially identical to a sequencein a sample nucleic acid (e.g., target sequence) or complement thereof.A “substantially identical” nucleotide sequence in an amplificationproduct will generally have a high degree of sequence identity to thenucleotide sequence being amplified or complement thereof (e.g., about75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than99% sequence identity), and variations sometimes are a result ofpolymerase infidelity or other variables.

In some embodiments, a nucleic acid amplification product comprises apolynucleotide that is continuously complementary to or substantiallyidentical to a target sequence in sample nucleic acid. Continuouslycomplementary generally refers to a nucleotide sequence in a firststrand, for example, where each base in order (e.g., read 5′ to 3′)pairs with a correspondingly ordered base in a second strand, and thereare no gaps, additional sequences or unpaired bases within the sequenceconsidered as continuously complementary. Stated another way,continuously complementary generally refers to all contiguous bases of anucleotide sequence in a first stand being complementary tocorresponding contiguous bases of a nucleotide sequence in a secondstrand. For example, a first strand having a sequence 5′-ATGCATGCATGC-3′(SEQ ID NO:10) would be considered as continuously complementary to asecond strand having a sequence 5′-GCATGCATGCAT-3′ (SEQ ID NO:11), whereall contiguous bases in the first strand are complementary to allcorresponding contiguous bases in the second strand. However, a firststrand having a sequence 5′-ATGCATAAAAAAGCATGC-3′ (SEQ ID NO:12) wouldnot be considered as continuously complementary to a second strandhaving a sequence 5′-GCATGCATGCAT-3′ (SEQ ID NO:11), because thesequence of six adenines (6 As) in the middle of the first strand wouldnot pair with bases in the second strand. A continuously complementarysequence sometimes is about 5 contiguous bases to about 25 contiguousbases in length, sometimes is about 6 contiguous bases to about 20contiguous bases in length, sometimes is about 7 contiguous bases toabout 18 contiguous bases in length, and sometimes is about 8 contiguousbases to about 16 contiguous bases in length. In some embodiments, anucleic acid amplification product consists of a polynucleotide that iscontinuously complementary to or substantially identical to a targetsequence in sample nucleic acid. Accordingly, in some embodiments, anucleic acid amplification product does not include any additionalsequences (e.g., at the 5′ and/or 3′ end, or within the product) thatare not continuously complementary to or substantially identical to atarget sequence, such as, for example, additional sequences incorporatedinto an amplification product by way of tailed primers or ligation,and/or additional sequences providing cleavage agent recognition sites(e.g., nicking enzyme recognition sites). Generally, unless a targetsequence comprises tandem repeats, an amplification product does notinclude product in the form of tandem repeats.

Nucleic acid amplification products may comprise sequences complementaryto or substantially identical to one or more primers used in anamplification reaction. In some embodiments, a nucleic acidamplification product comprises a first nucleotide sequence that iscontinuously complementary to or identical to a first primer sequence,and a second nucleotide sequence that is continuously complementary toor identical to a second primer sequence.

In some embodiments, nucleic acid amplification products comprise aspacer sequence. Generally, a spacer sequence in an amplificationproduct is a sequence (1 or more bases) continuously complementary to orsubstantially identical to a portion of a target sequence in the samplenucleic acid, and is flanked by sequences in the amplification productthat are complementary to or substantially identical to one or moreprimers used in an amplification reaction. A spacer sequence flanked bysequences in the amplification product generally lies between a firstsequence (complementary to or substantially identical to a first primer)and a second sequence (complementary to or substantially identical to asecond primer). Thus, an amplification product typically includes afirst sequence followed by a spacer sequences followed by a secondsequence. A spacer sequence generally is not complementary to orsubstantially identical to a sequence in the primer(s). In someembodiments, a spacer sequence comprises about 1 to 10 bases. Forexample, a spacer sequence may comprise 1 base, 2 bases, 3 bases, 4bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases. In someembodiments, a spacer sequence comprises about 1 to 5 bases.

In some embodiments, a nucleic acid amplification product consists of afirst nucleotide sequence that is continuously complementary to oridentical to a first primer sequence, a second nucleotide sequence thatis continuously complementary to or identical to a second primersequence, and a spacer sequence. Accordingly, in some embodiments, anucleic acid amplification product does not include any additionalsequences (e.g., at the 5′ and/or 3′ end; or within the product) thatare not continuously complementary to or identical to a first primersequence and a second primer sequence, and are not part of a spacersequence, such as, for example, additional sequences incorporated intoan amplification product by way of tailed or looped primers, ligation orother mechanism.

In some embodiments, a nucleic acid amplification product consistsessentially of a first nucleotide sequence that is continuouslycomplementary to or identical to a first primer sequence, a secondnucleotide sequence that is continuously complementary to or identicalto a second primer sequence, and a spacer sequence. Accordingly, in someembodiments, a nucleic acid amplification product generally does notinclude additional sequences (e.g., at the 5′ and/or 3′ end; or withinthe product) that are not continuously complementary to or identical toa first primer sequence and a second primer sequence, and are not partof a spacer sequence, such as, for example, additional sequencesincorporated into an amplification product by way of tailed or loopedprimers, ligation or other mechanism. However, in such embodiments, anucleic acid amplification product may include, for example, somemismatched (i.e., non-complementary) bases or one more extra bases(e.g., at the 5′ and/or 3′ end; or within the product) introduced intothe product by way of error or promiscuity in the amplification process.

Nucleic acid amplification products may be up to 50 bases in length. Insome embodiments, a nucleic acid amplification product is about 15 toabout 40 bases long. For example, a nucleic acid amplification productmay be 15 bases long, 16 bases long, 17 bases long, 18 bases long, 19bases long, 20 bases long, 21 bases long, 22 bases long, 23 bases long,24 bases long, 25 bases long, 26 bases long, 27 bases long, 28 baseslong, 29 bases long, 30 bases long, 31 bases long, 32 bases long, 33bases long, 34 bases long, 35 bases long, 36 bases long, 37 bases long,38 bases long, 39 bases long, or 40 bases long. In some embodiments, anamplification product is about 20 to about 40 bases long. In someembodiments, an amplification product is about 20 to about 30 baseslong. In some embodiments, nucleic acid amplification products for agiven target sequence have the same length or substantially the samelength (e.g., within 1 to 5 bases). Accordingly, nucleic acidamplification products for a given target sequence may produce a singlesignal (e.g., band on an electrophoresis gel) and generally do notproduce multiple signals indicative of multiple lengths (e.g., a ladderor smear on an electrophoresis gel). For multiplex reactions, nucleicacid amplification products for different target sequences may havedifferent lengths.

The methods and components described herein may be used for multiplexamplification. Multiplex amplification generally refers to theamplification of more than one nucleic acid of interest (e.g.,amplification or more than one target sequence). For example, multiplexamplification can refer to amplification of multiple sequences from thesame sample or amplification of one of several sequences in a sample.Multiplex amplification also may refer to amplification of one or moresequences present in multiple samples either simultaneously or instep-wise fashion. For example, a multiplex amplification may be usedfor amplifying least two target sequences that are capable of beingamplified (e.g., the amplification reaction comprises the appropriateprimers and enzymes to amplify at least two target sequences). In someinstances, an amplification reaction may be prepared to detect at leasttwo target sequences, but only one of the target sequences may bepresent in the sample being tested, such that both sequences are capableof being amplified, but only one sequence is amplified. In someinstances, where two target sequences are present, an amplificationreaction may result in the amplification of both target sequences. Amultiplex amplification reaction may result in the amplification of one,some, or all of the target sequences for which it comprises theappropriate primers and enzymes. In some instances, an amplificationreaction may be prepared to detect two sequences with one pair ofprimers, where one sequence is a target sequence and one sequence is acontrol sequence (e.g., a synthetic sequence capable of being amplifiedby the same primers as the target sequence and having a different spacerbase or sequence than the target). In some instances, an amplificationreaction may be prepared to detect multiple sets of sequences withcorresponding primer pairs, where each set includes a target sequenceand a control sequence.

Primers

Nucleic acid amplification generally is conducted in the presence of oneor more primers. A primer is generally characterized as anoligonucleotide that includes a nucleotide sequence capable ofhybridizing or annealing to a target nucleic acid, at or near (e.g.,adjacent to) a specific region of interest (i.e., target sequence).Primers can allow for specific determination of a target nucleic acidnucleotide sequence or detection of the target nucleic acid (e.g.,presence or absence of a sequence), or feature thereof, for example. Aprimer may be naturally occurring or synthetic. The term specific, orspecificity, generally refers to the binding or hybridization of onemolecule to another molecule, such as a primer for a targetpolynucleotide. That is, specific or specificity refers to therecognition, contact, and formation of a stable complex between twomolecules, as compared to substantially less recognition, contact, orcomplex formation of either of those two molecules with other molecules.The term anneal or hybridize generally refers to the formation of astable complex between two molecules. The terms primer, oligo, oroligonucleotide may be used interchangeably herein, when referring toprimers.

A primer may be designed and synthesized using suitable processes, andmay be of any length suitable for hybridizing to a target sequence andperforming an amplification process described herein. Primers often aredesigned according to a sequence in a target nucleic acid. A primer insome embodiments may be about 5 bases in length to about 30 bases inlength. For example, a primer may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 basesin length. In some embodiments, a primer is less than 28 bases inlength. In some embodiments, a primer is about 8 to about 16 bases inlength. In some embodiments, a primer is about 10 to about 12 bases inlength. A primer may be composed of naturally occurring and/ornon-naturally occurring nucleotides (e.g., modified nucleotides, labelednucleotides), or a mixture thereof. Primers suitable for use withmethods described herein may be synthesized and labeled using anysuitable technique. For example, primers may be chemically synthesizedaccording to the solid phase phosphoramidite triester method firstdescribed by Beaucage and Caruthers, Tetrahedron Letts., 22:1859-1862,1981, using an automated synthesizer, as described inNeedham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168, 1984.Purification of primers may be effected, for example, by nativeacrylamide gel electrophoresis or by anion-exchange high-performanceliquid chromatography (HPLC), for example, as described in Pearson andRegnier, J. Chrom., 255:137-149, 1983.

In some embodiments, a primer comprises modified nucleotides. In someembodiments, a primer consists essentially of modified nucleotides. Insome embodiments, a primer consists of modified nucleotides. Anucleotide (or base) may be modified according to any modificationdescribed herein or known in the art. Modifications may include thosemade during primer synthesis and/or may include post-syntheticmodifications. Modifications may include internal modifications,modifications and the 3′ end of a primer, and/or modifications at the 5′end of a primer. In some embodiments, a primer comprises a mixture ofmodified and unmodified nucleotides. In some embodiments, a primercomprises unmodified nucleotides. In some embodiments, a primer consistsessentially of unmodified nucleotides. In some embodiments, a primerconsists of unmodified nucleotides.

Modifications and modified bases may include, for example,phosphorylation, (e.g., 3′ phosphorylation, 5′ phosphorylation);attachment chemistry or linkers modifications (e.g., Acrydite™,adenylation, azide (NHS ester), digoxigenin (NHS ester),cholesteryl-TEG, I-Linker™ amino modifiers (e.g., amino modifier C6,amino modifier C12, amino modifier C6 dT, Uni-Link™ amino modifier),alkynes (e.g., 5′ hexynyl, 5-octadiynyl dU), biotinylation (e.g.,biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin,desthiobiotin-TEG), thiol modifications (e.g., thiol modifier C3 S-S,dithiol, thiol modifier C6 S-S)); fluorophores (e.g., Freedom™ Dyes,Alexa Fluor® Dyes, LI-COR IRDyes®, ATTO™ Dyes, Rhodamine Dyes, WellREDDyes, 6-FAM (azide), Texas Red®-X (NHS ester), Lightcycler® 640 (NHSester), Dy 750 (NHS ester)); Iowa Black® dark quenchers modifications(e.g., Iowa Black® FQ, Iowa Black® RQ); dark quenchers modifications(e.g., Black Hole Quencher®-1, Black Hole Quencher®-2, Dabcyl); spacers(C3 spacer, PC spacer, hexanediol, spacer 9, spacer 18,1′,2′-dideoxyribose (dSpacer); modified bases (e.g., 2-aminopurine,2,6-diaminopurine (2-amino-dA), 5-bromo dU, deoxyUridine, inverted dT,inverted dideoxy-T, dideoxy-C, 5-methyl dC, deoxylnosine, Super T®,Super G®, locked nucleic acids (LNA's), 5-nitroindole, 2′-O-methyl RNAbases, hydroxmethyl dC, UNA unlocked nucleic acid (e.g., UNA-A, UNA-U,UNA-C, UNA-G), Iso-dC, Iso-dG, Fluoro C, Fluoro U, Fluoro A, Fluoro G);phosphorothioate bonds modifications (e.g., phosphorothioated DNA bases,phosphorothioated RNA bases, phosphorothioated 2′ O-methyl bases,phosphorothioated LNA bases); and click chemistry modifications. In someembodiments, modifications and modified bases include uracil bases,ribonucleotide bases, O-methyl RNA bases, phosphorothioate linkages, 3′phosphate groups, spacer bases (such as C3 spacer or other spacerbases). For example, a primer may comprise one or more O-methyl RNAbases (e.g., 2′-O-methyl RNA bases). 2′-O-methyl RNA generally is apost-transcriptional modification of RNA found in tRNA and other smallRNAs. Primers can be directly synthesized that include 2′-O-methyl RNAbases. This modification can, for example, increase Tm of RNA:RNAduplexes and provide stability in the presence of single-strandedribonucleases and DNases. 2′-O-methyl RNA bases may be included inprimers, for example, to increase stability and binding affinity to atarget sequence. In some embodiments, a primer may comprise one or morephosphorothioate linkages (e.g., phosphorothioate bond modifications). Aphosphorothioate (PS) bond substitutes a sulfur atom for a non-bridgingoxygen in the phosphate backbone of a primer. This modificationtypically renders the internucleotide linkage resistant to nucleasedegradation. Phosphorothioate bonds may be introduced between about thelast 3 to 5 nucleotides at the 5′-end or the 3′-end of a primer toinhibit exonuclease degradation, for example. Phosphorothioate bondsincluded throughout an entire primer can help reduce attack byendonucleases, in certain instances. In some embodiments, a primer maycomprise a 3′ phosphate group. 3′ phosphorylation can inhibitdegradation by certain 3′-exonucleases and can be used to blockextension by DNA polymerases, in certain instances. In some embodiments,a primer may comprise one or more spacer bases (e.g., one or more C3spacers). A C3 spacer phosphoramidite can be incorporated internally orat the 5′-end of a primer. Multiple C3 spacers may be added at eitherend of a primer to introduce a long hydrophilic spacer arm for theattachment of fluorophores or other pendent groups, for example.

In some embodiments, a primer comprises DNA bases. In some embodiments,a primer comprises RNA bases. In some embodiments, a primer comprises amixture of DNA bases and RNA bases. DNA bases may be modified orunmodified. RNA bases may be modified or unmodified. In someembodiments, a primer consists essentially of DNA bases (e.g., modifiedDNA bases and/or unmodified DNA bases). In some embodiments, a primerconsists of DNA bases (e.g., modified DNA bases and/or unmodified DNAbases). In some embodiments, a primer consists essentially of unmodifiedDNA bases. In some embodiments, a primer consists of unmodified DNAbases. In some embodiments, a primer consists essentially of modifiedDNA bases. In some embodiments, a primer consists of modified DNA bases.In some embodiments, a primer consists essentially of RNA bases (e.g.,modified RNA bases and/or unmodified RNA bases). In some embodiments, aprimer consists of RNA bases (e.g., modified RNA bases and/or unmodifiedRNA bases). In some embodiments, a primer consists essentially ofunmodified RNA bases. In some embodiments, a primer consists ofunmodified RNA bases. In some embodiments, a primer consists essentiallyof modified RNA bases. In some embodiments, a primer consists ofmodified RNA bases.

In some embodiments, a primer comprises no RNA bases. In someembodiments, a primer comprises no RNA bases at the 3′ end. In someembodiments, a primer comprises a DNA base (or modified DNA base) at the3′ end. In some embodiments, a primer is not a chimeric primer. Achimeric primer is a primer comprising DNA and RNA bases. In someembodiments, a primer is a homogeneous primer. In some embodiments, aprimer is a homogeneous DNA primer. A homogeneous DNA primer maycomprise unmodified DNA bases, modified DNA bases, or a mixture ofmodified DNA bases and unmodified DNA bases, and generally do notinclude RNA bases.

In some embodiments, a primer comprises no cleavage agent recognitionsites. For example, a primer herein may comprise no nicking enzymerecognition sites. In some embodiments, a primer comprises no tail. Insome embodiments, a primer comprises no tail comprising a nicking enzymerecognition site.

All or a portion of a primer sequence may be complementary orsubstantially complementary to a target nucleic acid, in someembodiments. Substantially complementary with respect to sequencesgenerally refers to nucleotide sequences that will hybridize with eachother. The stringency of the hybridization conditions can be altered totolerate varying amounts of sequence mismatch. In some embodiments,target and primer sequences are at least 75% complementary to eachother. For example, target and primer sequences may be 75% or more, 76%or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% ormore, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more,87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more or 99% or more complementary to each other.

Primers that are substantially complimentary to a target nucleic acidsequence typically are also substantially identical to the compliment ofthe target nucleic acid sequence. That is, primers are substantiallyidentical to the anti-sense strand of the nucleic acid. Substantiallyidentical with respect to sequences generally refers to nucleotidesequences that are at least 75% identical to each other. For example,primers that are substantially identical to the anti-sense strand of atarget nucleic acid may 75% or more, 76% or more, 77% or more, 78% ormore, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more,84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more or 99% or moreidentical to each other. One test for determining whether two nucleotidesequences are substantially identical is to determine the percent ofidentical nucleotide sequences shared.

In some embodiments, primers comprise a pair of primers. A pair ofprimers may include a forward primer and a reverse primer (e.g., primersthat bind to the sense and antisense strands of a target nucleic acid).In some embodiments, primers consist of a pair of primers (i.e. aforward primer and a reverse primer). Accordingly, in some embodiments,amplification of a target sequence is performed using a pair of primersand no additional primers or oligonucleotides are included in theamplification of the target sequence (e.g., the amplification reactioncomponents comprise no additional primer pairs for a given targetsequence, no nested primers, no bumper primers, no oligonucleotidesother than the primers, no probes, and the like). In some embodiments,primers consist of a pair of primers, however, in certain instances, anamplification reaction may include additional primer pairs foramplifying different target sequences, such as in a multiplexamplification. In some embodiments, primers consist of a pair ofprimers, however, in certain instances, an amplification reaction mayinclude additional primers, oligonucleotides or probes for a detectionprocess that are not considered part of amplification.

In some embodiments primers are used in sets. An amplification primerset may include a pair of forward and reverse primers for a given targetsequence. For multiplex amplification, primers that amplify a firsttarget sequence are considered a primer set, and primers that amplify asecond target sequence are considered a different primer set.

In some embodiments, amplification reaction components comprise a firstprimer (first oligonucleotide) complementary to a target sequence in afirst strand (e.g., sense strand, forward strand) of a sample nucleicacid, and a second primer (second oligonucleotide) complementary to atarget sequence in a second strand (e.g., antisense strand, reversestrand) of a sample nucleic acid. In some embodiments, a first primer(first oligonucleotide) comprises a first polynucleotide continuouslycomplementary to a target sequence in a first strand of sample nucleicacid, and a second primer (second oligonucleotide) comprises a secondpolynucleotide continuously complementary to a target sequence in asecond strand of sample nucleic acid. Continuously complementary for aprimer-target generally refers to a nucleotide sequence in a primer,where each base in order pairs with a correspondingly ordered base in atarget sequence, and there are no gaps, additional sequences or unpairedbases within the sequence considered as continuously complementary.Stated another way, continuously complementary generally refers to allcontiguous bases of a nucleotide sequence in a primer beingcomplementary to corresponding contiguous bases of a nucleotide sequencein a target.

In some embodiments, a first primer (first oligonucleotide) consists ofa first polynucleotide continuously complementary to a target sequencein a first strand of sample nucleic acid, and a second primer (secondoligonucleotide) consists of a second polynucleotide continuouslycomplementary to a target sequence in the second strand of the samplenucleic acid. Accordingly, in some embodiments, a primer does notinclude any additional sequences (e.g., at the 5′ and/or 3′ end, orwithin the primer) that are not continuously complementary to a targetsequence, such as, for example, additional sequences present in tailedprimers or looped primers, and/or additional sequences providingcleavage agent recognition sites (e.g., nicking enzyme recognitionsites). In some embodiments, amplification reaction components do notcomprise primers comprising additional sequences (i.e., sequences otherthan the sequence that is continuously complementary to a targetsequence) such as, for example, tailed primers, looped primers, primerscapable of forming step-loop structures, hairpin structures, and/oradditional sequences providing cleavage agent recognition sites (e.g.,nicking enzyme recognition sites), and the like.

In some embodiments, a first primer (first oligonucleotide) consistsessentially of a first polynucleotide continuously complementary to atarget sequence in a first strand of sample nucleic acid, and a secondprimer (second oligonucleotide) consists essentially of a secondpolynucleotide continuously complementary to a target sequence in thesecond strand of the sample nucleic acid. Accordingly, in someembodiments, a primer generally does not include any additionalsequences (e.g., at the 5′ and/or 3′ end, or within the primer) that arenot continuously complementary to a target sequence, such as, forexample, additional sequences present in tailed primers or loopedprimers, and/or additional sequences providing cleavage agentrecognition sites (e.g., nicking enzyme recognition sites). However, insuch embodiments, a primer may include one or more additional bases(e.g., at the 5′ and/or 3′ end, or within the primer) that do not add afunctional feature to the primer. For example, additional sequencespresent in tailed primers or looped primers generally add a functionalfeature and would be excluded from primers in such embodiments.

A primer, in certain embodiments, may contain a modification such as oneor more inosines, abasic sites, locked nucleic acids, minor groovebinders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiersor any modifier that changes the binding properties of the primer. Aprimer, in certain embodiments, may contain a detectable molecule orentity (e.g., a fluorophore, radioisotope, colorimetric agent, particle,enzyme and the like).

Polymerase

In some embodiments, amplification reaction components comprise one ormore polymerases. Polymerases are proteins capable of catalyzing thespecific incorporation of nucleotides to extend a 3′ hydroxyl terminusof a primer molecule, such as, for example, an amplification primerdescribed herein, against a nucleic acid target sequence (e.g., to whicha primer is annealed). Polymerases may include, for example,thermophilic or hyperthermophilic polymerases that can have activity atan elevated reaction temperature (e.g., above 55 degrees Celsius, above60 degrees Celsius, above 65 degrees Celsius, above 70 degrees Celsius,above 75 degrees Celsius, above 80 degrees Celsius, above 85 degreesCelsius, above 90 degrees Celsius, above 95 degrees Celsius, above 100degrees Celsius). A hyperthermophilic polymerase may be referred to as ahyperthermophile polymerase. A polymerase having hyperthermophilicpolymerase activity may be referred to as having hyperthermophilepolymerase activity. A polymerase may or may not have stranddisplacement capabilities. In some embodiments, a polymerase canincorporate about 1 to about 50 nucleotides in a single synthesis. Forexample, a polymerase may incorporate about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in a single synthesis. In some embodiments, apolymerase, can incorporate 20 to 40 nucleotides in a single synthesis.In some embodiments, a polymerase, can incorporate up to 50 nucleotidesin a single synthesis. In some embodiments, a polymerase, canincorporate up to 40 nucleotides in a single synthesis. In someembodiments, a polymerase, can incorporate up to 30 nucleotides in asingle synthesis. In some embodiments, a polymerase, can incorporate upto 20 nucleotides in a single synthesis.

In some embodiments, amplification reaction components comprise one ormore DNA polymerases. In some embodiments, amplification reactioncomponents comprise one or more DNA polymerases selected from thefollowing: 9° N DNA polymerase; 9° Nm™ DNA polymerase; Therminator™ DNAPolymerase; Therminator™ II DNA Polymerase; Therminator™ III DNAPolymerase; Therminator™ γ DNA Polymerase; Bst DNA polymerase; Bst DNApolymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E.coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3′-5′exo-); T4 DNA polymerase; T7 DNA polymerase; Deep VentR™ (exo-) DNAPolymerase; Deep VentR™ DNA Polymerase; DyNAzyme™ EXT DNA; DyNAzyme™ IIHot Start DNA Polymerase; Phusion™ High-Fidelity DNA Polymerase; VentR®DNA Polymerase; VentR® (exo-) DNA Polymerase; RepliPHI™ Phi29 DNAPolymerase; rBst DNA Polymerase, large fragment (IsoTherm™ DNAPolymerase); MasterAmp™ AmpliTherm™ DNA Polymerase; Tag DNA polymerase;Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNApolymerase; Tbr DNA polymerase; DNA polymerase Beta; and ThermoPhi DNApolymerase.

In some embodiments, amplification reaction components comprise one ormore hyperthermophile DNA polymerases. Generally, hyperthermophile DNApolymerases are thermostable at high temperatures. For example, ahyperthermophile DNA polymerase may have a half-life of about 5 to 10hours at 95 degrees Celsius and a half-life of about 1 to 3 hours at 100degrees Celsius. In some embodiments, amplification reaction componentscomprise one or more hyperthermophile DNA polymerases from Archaea. Insome embodiments, amplification reaction components comprise one or morehyperthermophile DNA polymerases from Thermococcus. In some embodiments,amplification reaction components comprise one or more hyperthermophileDNA polymerases from Thermococcaceaen archaean. In some embodiments,amplification reaction components comprise one or more hyperthermophileDNA polymerases from Pyrococcus. In some embodiments, amplificationreaction components comprise one or more hyperthermophile DNApolymerases from Methanococcaceae. In some embodiments, amplificationreaction components comprise one or more hyperthermophile DNApolymerases from Methanococcus. In some embodiments, amplificationreaction components comprise one or more hyperthermophile DNApolymerases from Thermus. In some embodiments, amplification reactioncomponents comprise one or more hyperthermophile DNA polymerases fromThermus thermophiles.

In some embodiments, amplification reaction components comprise ahyperthermophile DNA polymerase or functional fragment thereof. Afunctional fragment generally retains one or more functions of afull-length polymerase such as, for example, the capability topolymerize DNA (e.g., in an amplification reaction). In some instances,a functional fragment performs a function (e.g., polymerization of DNAin an amplification reaction) at a level that is at least about 50% thelevel of function for a full length polymerase. In some instances, afunctional fragment performs a function (e.g., polymerization of DNA inan amplification reaction) at a level that is at least about 75% thelevel of function for a full length polymerase. In some instances, afunctional fragment performs a function (e.g., polymerization of DNA inan amplification reaction) at a level that is at least about 90% thelevel of function for a full length polymerase. In some instances, afunctional fragment performs a function (e.g., polymerization of DNA inan amplification reaction) at a level that is at least about 95% thelevel of function for a full length polymerase. Levels of polymeraseactivity can be assessed, for example, using a detectable nucleic acidamplification method, such as, for example, a detectable nucleic acidamplification method described herein. In some embodiments,amplification reaction components comprise a hyperthermophile DNApolymerase comprising an amino acid sequence of SEQ ID NO:8 or afunctional fragment of SEQ ID NO:8. In some embodiments, amplificationreaction components comprise a hyperthermophile DNA polymerasecomprising an amino acid sequence of SEQ ID NO:9 or a functionalfragment of SEQ ID NO:9.

In some embodiments, amplification reaction components comprise apolymerase comprising an amino acid sequence that is at least about 90%identical to a hyperthermophile polymerase or a functional fragmentthereof (i.e., a functional fragment as described herein of a polymerasecomprising an amino acid sequence that is at least about 90% identicalto a hyperthermophile polymerase). The degree of sequence identity canbe determined, for example, by performing an amino acid sequencealignment. In some embodiments, amplification reaction componentscomprise a polymerase comprising an amino acid sequence that is at leastabout 90% identical to the amino acid sequence of SEQ ID NO:8 or afunctional fragment thereof (i.e., a functional fragment as describedherein of a polymerase comprising an amino acid sequence that is atleast about 90% identical to SEQ ID NO:8). In some embodiments,amplification reaction components comprise a polymerase comprising anamino acid sequence that is at least about 95% identical to the aminoacid sequence of SEQ ID NO:8 or a functional fragment thereof. In someembodiments, amplification reaction components comprise a polymerasecomprising an amino acid sequence that is at least about 99% identicalto the amino acid sequence of SEQ ID NO:8 or a functional fragmentthereof. In some embodiments, amplification reaction components comprisea polymerase comprising an amino acid sequence that is at least about90% identical to the amino acid sequence of SEQ ID NO:9 or a functionalfragment thereof (i.e., a functional fragment as described herein of apolymerase comprising an amino acid sequence that is at least about 90%identical to SEQ ID NO:9). In some embodiments, amplification reactioncomponents comprise a polymerase comprising an amino acid sequence thatis at least about 95% identical to the amino acid sequence of SEQ IDNO:9 or a functional fragment thereof. In some embodiments,amplification reaction components comprise a polymerase comprising anamino acid sequence that is at least about 99% identical to the aminoacid sequence of SEQ ID NO:9 or a functional fragment thereof.

In some embodiments, a polymerase may possess reverse transcriptioncapabilities. In such instances, an amplification reaction can amplifyRNA targets, for example, in a single step without the use of a separatereverse transcriptase. Non-limiting examples of polymerases that possessreverse transcriptase capabilities include Bst (large fragment), 9° NDNA polymerase, 9° Nm™ DNA polymerase, Therminator™, Therminator™ II,and the like). In some embodiments, amplification reaction componentscomprise one or more separate reverse transcriptases. In someembodiments, more than one polymerase may be included in in anamplification reaction. For example, an amplification reaction maycomprise a polymerase having reverse transcriptase activity and a secondpolymerase having no reverse transcriptase activity.

In some embodiments, one or more polymerases having exonuclease activityare used during amplification. In some embodiments, one or morepolymerases having no exonuclease activity are used duringamplification. In some embodiments, one or more polymerases having lowexonuclease activity are used during amplification. In certaininstances, a polymerase having no or low exonuclease activity comprisesone or more modifications (e.g., amino acid substitutions) that reduceor eliminate the exonuclease activity of the polymerase. A modifiedpolymerase having low exonuclease activity may have 10% or lessexonuclease activity compared to an unmodified polymerase. For example,a modified polymerase having low exonuclease activity may have less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% exonuclease activitycompared to an unmodified polymerase. In some embodiments, a polymerasehas no or low 5′ to 3′ exonuclease activity. In some embodiments, apolymerase has no or low 3′ to 5′ exonuclease activity. In someembodiments, a polymerase has no or low single strand dependentexonuclease activity. In some embodiments, a polymerase has no or lowdouble strand dependent exonuclease activity. Non limiting examples ofcertain modifications that can reduce or eliminate exonuclease activityfor a polymerase include one or more amino acid substitutions atposition 141 and/or 143 and/or 458 of SEQ ID NO:8, or at a positioncorresponding to position 141 and/or 143 and/or 458 of SEQ ID NO:8. Anamino acid position corresponding to a position in SEQ ID NO:8 may beidentified, for example, by performing an amino acid sequence alignment.In some instances, modification(s) include a substitution of the nativeamino acid at position 141 to an alanine. In some instances themodification(s) include D141A. In some instances, modification(s)include a substitution of the native amino acid at position 143 to analanine. In some instances the modification(s) include E143A. In someinstances, modification(s) include a substitution of the native aminoacid at position 143 to an aspartate. In some instances themodification(s) include E143D. In some instances, modification(s)include a substitution of the native amino acid at position 485 to aleucine. In some instances the modification(s) include A485L. In someinstances, the modifications include D141A, E143A and A485L.

Detection and Quantification

The methods described herein may further comprise detecting and/orquantifying a nucleic acid amplification product. An amplificationproduct may be detected and/or quantified by any suitable detectionand/or quantification method including, for example, any detectionmethod or quantification method described herein. Non-limiting examplesof detection and/or quantification methods include molecular beacon(e.g., real-time, endpoint), lateral flow, fluorescence resonance energytransfer (FRET), fluorescence polarization (FP), surface capture, 5′ to3′ exonuclease hydrolysis probes (e.g., TAQMAN), intercalating/bindingdyes, absorbance methods (e.g., colorimetric, turbidity),electrophoresis (e.g., gel electrophoresis, capillary electrophoresis),mass spectrometry, nucleic acid sequencing, digital amplification, aprimer extension method (e.g., iPLEX™), Molecular Inversion Probe (MIP)technology from Affymetrix, restriction fragment length polymorphism(RFLP analysis), allele specific oligonucleotide (ASO) analysis,methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprimeanalysis, Reverse dot blot, GeneChip microarrays, Dynamicallele-specific hybridization (DASH), Peptide nucleic acid (PNA) andlocked nucleic acids (LNA) probes, AlphaScreen, SNPstream, genetic bitanalysis (GBA), Multiplex minisequencing, SNaPshot, GOOD assay,Microarray miniseq, arrayed primer extension (APEX), Microarray primerextension, Tag arrays, Coded microspheres, Template-directedincorporation (TDI), colorimetric oligonucleotide ligation assay (OLA),sequence-coded OLA, microarray ligation, ligase chain reaction, padlockprobes, invader assay, hybridization using at least one probe,hybridization using at least one fluorescently labeled probe, cloningand sequencing, the use of hybridization probes and quantitative realtime polymerase chain reaction (QRT-PCR), nanopore sequencing, chips andcombinations thereof. In some embodiments, detecting a nucleic acidamplification product comprises use of a real-time detection method(i.e., product is detected and/or continuously monitored during anamplification process). In some embodiments, detecting a nucleic acidamplification product comprises use of an endpoint detection method(i.e., product is detected after completing or stopping an amplificationprocess). Nucleic acid detection methods may also employ the use oflabeled nucleotides incorporated directly into a target sequence or intoprobes containing complementary sequences to a target. Such labels maybe radioactive and/or fluorescent in nature and can be resolved in anyof the manners discussed herein. In some embodiments, quantification ofa nucleic acid amplification product may be achieved using certaindetection methods described below. In certain instances, a detectionmethod may be used in conjunction with a measurement of signalintensity, and/or generation of (or reference to) a standard curveand/or look-up table for quantification of a nucleic acid amplificationproduct.

In some embodiments, detecting a nucleic acid amplification productcomprises use of molecular beacon technology. The term molecular beacongenerally refers to a detectable molecule, where the detectable propertyof the molecule is detectable under certain conditions, thereby enablingthe molecule to function as a specific and informative signal.Non-limiting examples of detectable properties include, opticalproperties (e.g., fluorescence), electrical properties, magneticproperties, chemical properties and time or speed through an opening ofknown size. Molecular beacons for detecting nucleic acid molecules maybe, for example, hair-pin shaped oligonucleotides containing afluorophore on one end and a quenching dye on the opposite end. The loopof the hair-pin may contain a probe sequence that is complementary to atarget sequence and the stem is formed by annealing of complementary armsequences located on either side of the probe sequence. A fluorophoreand a quenching molecule can be covalently linked at opposite ends ofeach arm. Under conditions that prevent the oligonucleotides fromhybridizing to its complementary target or when the molecular beacon isfree in solution, the fluorescent and quenching molecules are proximalto one another preventing fluorescence resonance energy transfer (FRET).When the molecular beacon encounters a target molecule (e.g., a nucleicacid amplification product), hybridization can occur, and the loopstructure is converted to a stable more rigid conformation causingseparation of the fluorophore and quencher molecules leading tofluorescence (Tyagi et al. Nature Biotechnology 14: March 1996,303-308). Due to the specificity of the probe, the generation offluorescence generally is exclusively due to the synthesis of theintended amplified product. In some instances, a molecular beacon probesequence hybridizes to a sequence in an amplification product that isidentical to or complementary to a sequence in a target nucleic acid. Insome instances, a molecular beacon probe sequence hybridizes to asequence in an amplification product that is not identical to orcomplementary to a sequence in a target nucleic acid (e.g., hybridizesto a sequence added to an amplification product by way of a tailedamplification primer or ligation).

Molecular beacons are highly specific and can discern a singlenucleotide polymorphism. Molecular beacons also can be synthesized withdifferent colored fluorophores and different target sequences, enablingsimultaneous detection of several products in the same reaction (e.g.,in a multiplex reaction). For quantitative amplification processes,molecular beacons can specifically bind to the amplified targetfollowing each cycle of amplification, and because non-hybridizedmolecular beacons are dark, it is not necessary to isolate theprobe-target hybrids to quantitatively determine the amount of amplifiedproduct. The resulting signal is proportional to the amount of amplifiedproduct. Detection using molecular beacons can be done in real time oras an end-point detection method. In some instances, certain reactionconditions may be optimized for each primer/probe set to ensure accuracyand precision.

In some embodiments, detecting a nucleic acid amplification productcomprises use of lateral flow. Use of lateral flow typically includesuse of a lateral flow device. These devices generally include a solidphase fluid permeable flow path through which fluid flows through bycapillary force. Example devices include, but are not limited to,dipstick assays and thin layer chromatographic plates with variousappropriate coatings. Immobilized on the flow path are various bindingreagents for the sample, binding partners or conjugates involvingbinding partners for the sample and signal producing systems. Detectioncan be achieved in several manners including, for example, enzymaticdetection, nanoparticle detection, colorimetric detection, andfluorescence detection. Enzymatic detection may involve enzyme-labeledprobes that are hybridized to complementary nucleic aid targets on thesurface of the lateral flow device. The resulting complex can be treatedwith appropriate markers to develop a readable signal. Nanoparticledetection involves bead technology that may use colloidal gold, latexand/or paramagnetic nanoparticles. In one example, beads may beconjugated to an anti-biotin antibody. Target sequences may be directlybiotinylated, or target sequences may be hybridized to sequence-specificbiotinylated probes. Gold and latex give rise to colorimetric signalsvisible to the naked eye, and paramagnetic particles give rise to anon-visual signal when excited in a magnetic field and can beinterpreted by a specialized reader. Fluorescence-based lateral flowdetection methods also may be used and include, for example, dualfluorescein and biotin-labeled oligo probe methods, UPT-N ALP utilizingup-converting phosphor reporters composed of lanthanide elementsembedded in a crystal (Corstjens et al., Clinical Chemistry, 47:10,1885-1893, 2001), and the use of quantum dots.

Nucleic acids may be captured on lateral flow devices. Means of capturemay include antibody-dependent and antibody-independent methods.Antibody-dependent capture generally comprises an antibody capture lineand a labeled probe of complementary sequence to the target.Antibody-independent capture generally uses non-covalent interactionsbetween two binding partners, for example, the high affinity andirreversible linkage between a biotinylated probe and a streptavidinline. Capture probes may be immobilized directly on lateral flowmembranes. Both antibody-dependent and antibody-independent methods maybe used, for example, for detecting amplification products generated ina multiplex reaction.

In some embodiments, detecting a nucleic acid amplification productcomprises use of fluorescence resonance energy transfer (FRET). FRET isan energy transfer mechanism between two chromophores: a donor and anacceptor molecule. Briefly, a donor fluorophore molecule is excited at aspecific excitation wavelength. The subsequent emission from the donormolecule as it returns to its ground state may transfer excitationenergy to the acceptor molecule through a long range dipole-dipoleinteraction. The emission intensity of the acceptor molecule can bemonitored and is a function of the distance between the donor and theacceptor, the overlap of the donor emission spectrum and the acceptorabsorption spectrum and the orientation of the donor emission dipolemoment and the acceptor absorption dipole moment. FRET can be useful forquantifying molecular dynamics, for example, in DNA-DNA interactions asdescribed for molecular beacons. For monitoring the production of aspecific product, a probe can be labeled with a donor molecule on oneend and an acceptor molecule on the other. Probe-target hybridizationbrings a change in the distance or orientation of the donor and acceptorand FRET change is observed.

In some embodiments, detecting a nucleic acid amplification productcomprises use of fluorescence polarization (FP). Fluorescencepolarization techniques generally are based on the principle that afluorescently labeled compound when excited by linearly polarized lightwill emit fluorescence having a degree of polarization inversely relatedto its rate of rotation. Therefore, when a molecule such as atracer-nucleic acid conjugate, for example, having a fluorescent labelis excited with linearly polarized light, the emitted light remainshighly polarized because the fluorophore is constrained from rotatingbetween the time light is absorbed and emitted. When a free tracercompound (i.e., unbound to a nucleic acid) is excited by linearlypolarized light, its rotation is much faster than the correspondingtracer-nucleic acid conjugate and the molecules are more randomlyoriented, therefore, the emitted light is depolarized. Thus,fluorescence polarization provides a quantitative means for measuringthe amount of tracer-nucleic acid conjugate produced in an amplificationreaction.

In some embodiments, detecting a nucleic acid amplification productcomprises use of surface capture. This may be accomplished by theimmobilization of specific oligonucleotides to a surface producing abiosensor that is both highly sensitive and selective. Example surfacesthat may be used include gold and carbon, and a surface capture methodmay use a number of covalent or noncovalent coupling methods to attach aprobe to the surface. The subsequent detection of a target nucleic acidcan be monitored by a variety of methods.

In some embodiments, detecting a nucleic acid amplification productcomprises use of 5′ to 3′ exonuclease hydrolysis probes (e.g., TAQMAN).TAQMAN probes, for example, are hydrolysis probes that can increase thespecificity of a quantitative amplification method (e.g., quantitativePCR). The TAQMAN probe principle relies on 1) the 5′ to 3′ exonucleaseactivity of Taq polymerase to cleave a dual-labeled probe duringhybridization to a complementary target sequence and 2)fluorophore-based detection. A resulting fluorescence signal permitsquantitative measurements of the accumulation of amplification productduring the exponential stages of amplification, and the TAQMAN probe cansignificantly increase the specificity of the detection.

In some embodiments, detecting a nucleic acid amplification productcomprises use of intercalating and/or binding dyes. In some embodiments,detecting a nucleic acid amplification product comprises use of dyesthat specifically stain nucleic acid. For example, intercalating dyesexhibit enhanced fluorescence upon binding to DNA or RNA. Dyes mayinclude DNA or RNA intercalating fluorophores and may include forexample, SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes,PicoGreen®, propidium iodide, SYBR® I (an asymmetrical cyanine dye),SYBR® II, TOTO (a thiaxole orange dimer) and YOYO (an oxazole yellowdimer). Dyes provide an opportunity for increasing the sensitivity ofnucleic acid detection when used in conjunction with various detectionmethods. For example, ethidium bromide may be used for staining DNA inagarose gels after gel electrophoresis; propidium iodide and Hoechst33258 may be used in flow cytometry to determine DNA ploidy of cells;SYBR® Green 1 may be used in the analysis of double-stranded DNA bycapillary electrophoresis with laser induced fluorescence detection; andPicoGreen® may be used to enhance the detection of double-stranded DNAafter matched ion pair polynucleotide chromatography.

In some embodiments, detecting a nucleic acid amplification productcomprises use of absorbance methods (e.g., colorimetric, turbidity). Insome instances, detection and/or quantitation of nucleic acid can beachieved by directly converting absorbance (e.g., UV absorbancemeasurements at 260 nm) to concentration, for example. Directmeasurement of nucleic acid can be converted to concentration using theBeer Lambert law which relates absorbance to concentration using thepath length of the measurement and an extinction coefficient. In someembodiments, detecting a nucleic acid amplification product comprisesuse of a colorimetric detection method. Any suitable colorimetricdetection may be used, and non-limiting examples include assays that usenanoparticles (e.g., metallic nanoparticles, modified nanoparticles,unmodified nanoparticles) and/or peptide nucleic acid (PNA) probes. Forexample, certain gold nanoparticle-based methods typically rely on aquantitative coupling between target recognition and the aggregation ofthe nanoparticles, which, in turn, can lead to a change in the photonicproperties (e.g., color) of a nanoparticle solution.

In some embodiments, detecting a nucleic acid amplification productcomprises use of electrophoresis (e.g., gel electrophoresis, capillaryelectrophoresis). Gel electrophoresis involves the separation of nucleicacids through a matrix, generally a cross-linked polymer, using anelectromotive force that pulls the molecules through the matrix.Molecules move through the matrix at different rates causing aseparation between products that can be visualized and interpreted via anumber of methods including but not limited to; autoradiography,phosphorimaging, and staining with nucleic acid chelating dyes.Capillary-gel electrophoresis (CGE) is a combination of traditional gelelectrophoresis and liquid chromatography that employs a medium such aspolyacrylamide in a narrow bore capillary to generate fast,high-efficient separations of nucleic acid molecules with up to singlebase resolution. CGE may be combined with laser induced fluorescence(LIF) detection where as few as six molecules of stained DNA can bedetected. CGE/LIF detection generally involves the use of fluorescentDNA intercalating dyes including ethidium bromide, YOYO and SYBR® Green1, and also may involve the use of fluorescent DNA derivatives wherefluorescent dye is covalently bound to DNA. Simultaneous identificationof several different target sequences (e.g., products from a multiplexreaction) may be made using this method.

In some embodiments, detecting a nucleic acid amplification productcomprises use of mass spectrometry. Mass Spectrometry is an analyticaltechnique that may be used to determine the structure and quantity of anucleic acid and can be used to provide rapid analysis of complexmixtures. Following amplification, samples can be ionized, the resultingions separated in electric and/or magnetic fields according to theirmass-to-charge ratio, and a detector measures the mass-to-charge ratioof ions (Crain, P. F. and McCloskey, J. A., Current Opinion inBiotechnology 9: 25-34 (1998)). Mass spectrometry methods include, forexample, MALDI, MALDI-TOF, or Electrospray. These methods may becombined with gas chromatography (GC/MS) and liquid chromatography(LC/MS). Mass spectrometry (e.g., matrix-assisted laserdesorption/ionization mass spectrometry (MALDI MS)) can be highthroughput due to high-speed signal acquisition and automated analysisoff solid surfaces.

In some embodiments, detecting a nucleic acid amplification productcomprises use of nucleic acid sequencing. The entire sequence or apartial sequence of an amplification product may be determined, and thedetermined nucleotide sequence may be referred to as a read. Forexample, linear amplification products may be analyzed directly withoutfurther amplification in some embodiments (e.g., by usingsingle-molecule sequencing methodology). In certain embodiments, linearamplification products may be subject to further amplification and thenanalyzed (e.g., using sequencing by ligation or pyrosequencingmethodology). Reads may be subject to different types of sequenceanalysis. Any suitable sequencing method can be utilized to detect, andin some instances determine the amount of, detectable products generatedby the amplification methods described herein. Non-limiting examples ofsequencing methods include single-end sequencing, paired-end sequencing,reversible terminator-based sequencing, sequencing by ligation,pyrosequencing, sequencing by synthesis, single-molecule sequencing,multiplex sequencing, solid phase single nucleotide sequencing, andnanopore sequencing.

In some embodiments, detecting a nucleic acid amplification productcomprises use of digital amplification (e.g., digital PCR). Digital PCR,for example, takes advantage of nucleic acid (DNA, cDNA or RNA)amplification on a single molecule level, and offers a highly sensitivemethod for quantifying low copy number nucleic acid. Systems for digitalamplification and analysis of nucleic acids are available (e.g.,Fluidigm® Corporation).

Kits

Kits of may comprise, for example, one or more polymerases and one ormore primers, and optionally one or more reverse transcriptases, asdescribed herein. Where one target is amplified, a pair of primers(forward and reverse) may be included in the kit. Where multiple targetsequences are amplified, a plurality of primer pairs may be included inthe kit. A kit may include a control polynucleotide, and where multipletarget sequences are amplified, a plurality of control polynucleotidesmay be included in the kit.

Kits may also comprise one or more of the components in any number ofseparate vessels, chambers, containers, packets, tubes, vials,microtiter plates and the like, or the components may be combined invarious combinations in such containers. Components of the kit may, forexample, be present in one or more containers. In some embodiments, allof the components are provided in one container. In some embodiments,the enzymes (e.g., polymerase(s) and/or reverse transcriptase(s)) may beprovided in a separate container from the primers. The components may,for example, be lyophilized, freeze dried, or in a stable buffer. In oneexample, polymerase(s) and/or reverse transcriptase(s) are inlyophilized form in a single container, and the primers are eitherlyophilized, freeze dried, or in buffer, in a different container. Insome embodiments, polymerase(s) and/or reverse transcriptase(s), and theprimers are, in lyophilized form, in a single container.

Kits may further comprise, for example, dNTPs used in the reaction, ormodified nucleotides, vessels, cuvettes or other containers used for thereaction, or a vial of water or buffer for re-hydrating lyophilizedcomponents. The buffer used may, for example, be appropriate for bothpolymerase and primer annealing activity.

Kits may also comprise instructions for performing one or more methodsdescribed herein and/or a description of one or more componentsdescribed herein. Instructions and/or descriptions may be in printedform and may be included in a kit insert. A kit also may include awritten description of an internet location that provides suchinstructions or descriptions.

Kits may further comprise reagents used for detection methods, such as,for example, reagents used for FRET, lateral flow devices, dipsticks,fluorescent dye, colloidal gold particles, latex particles, a molecularbeacon, or polystyrene beads.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology.

Example 1: Detection of Chlamydia trachomatis by IsothermalAmplification Technology

In this example, the detection of nucleic acid from Chlamydiatrachomatis is performed using isothermal amplification technology.

Real-Time Detection of Chlamydia Genomic DNA

A real-time assay for detection of chlamydia genomic DNA withfluorescent DNA dye was tested. In this assay, SYTO 82 was used, whichis an orange fluorescent nucleic acid stain that exhibits bright orangefluorescence upon binding to nucleic acids. Master mix solutions wereprepared with 20 mM Tris-HCl pH 8.8 at 25° C., 10 mM (NH₄)₂SO₄, 10 mMKCl, 4 mM MgSO₄, 0.1% Triton® X-100, 1 mM DTT, 2 μM SYTO 82, 0.25 mMdNTP, and 1 unit per reaction of modified 9 Degrees North (9° Nm™) DNApolymerase (New England BioLabs, Ipswich, Mass.). The amino acidsequence of 9° Nm™ DNA polymerase is set forth herein as SEQ ID NO:9. Aprimer set targeting a specific sequence within the 7,500 base pair C.trachomatis cryptic plasmid DNA was used, which included an 11nucleotide forward primer (i.e., Ct_F11: 5′-GGCTTATGGAG-3′ (SEQ IDNO:1)) and a 10 nucleotide reverse primer (i.e., Ct_R10:5′-ATACCGCTTA-3′ (SEQ ID NO:2)). The assay was designed to generate 22base DNA products which include a one base spacer. The spacer is anucleotide between the 3′ ends of the primers, and this nucleotide isnot present in either of the primer sequences. The primers were eachused at a final concentration 500 nM, and were mixed with either 2000copies of chlamydia genomic DNA or Tris-EDTA buffer (TE) as a no targetcontrol (NTC). In certain instances, dH₂O was used as a NTC. The primermixtures were placed in separate reaction wells from the master mixsolutions. The assay components were incubated at 65° C. for 2 minutes,and then combined to initiate the isothermal reaction. The results ofthe isothermal amplification reaction are presented in FIG. 1.

Electrospray Ionization Mass Spectrometry (ESI-MS) Confirmation ofSpecific Product Generation from Isothermal Amplification Reactions

Amplification products generated from the isothermal amplificationreactions described above were tested by Electrospray Ionization MassSpectrometry (ESI-MS) to verify the specificity of the assay. Theprimers described above were each used at a final concentration 500 nM,and were mixed with either 20,000 copies of chlamydia genomic DNA orTris-EDTA buffer (TE) as a no target control (NTC). In certaininstances, dH₂O was used as a NTC. The chlamydia genomic DNA wasamplified at 65° C. for 10 minutes under the conditions described above.After amplification, the reactions were inactivated with Tris-EGTA(final 20 mM Tris-EGTA, pH8.5). The reactions were then desalted andlyophilized before ESI-MS analysis.

As shown in FIG. 2, ESI-MS results confirmed that the dominant productsfrom the isothermal amplification of genomic DNA were specific 22-baseproducts (i.e., 22-base forward product and 22-base reverse product). Incomparison, the NTC reaction generated non-specific products with muchless intensity than those with specific products.

Limit of Detection (LOD) for Chlamydia Genomic DNA Detection

Sensitivity of chlamydia genomic DNA detection was tested using anisothermal amplification assay. Under this approach, a 10 nucleotideprimer assay under asymmetric amplification conditions was used forendpoint molecular beacon detection. A master mix solution was preparedwith 20 mM Tris-HCl pH 8.8 at 25° C., 10 mM (NH₄)₂SO₄, 10 mM KCl, 4 mMMgSO₄, 0.1% Triton® X-100, 1 mM DTT, 0.25 mM dNTP, and 1 unit perreaction of modified 9 Degrees North (9° Nm™) DNA polymerase (NewEngland BioLabs, Ipswich, Mass.). A primer set targeting a specificsequence within the 7,500 base pair C. trachomatis cryptic plasmid DNAwas used, which included a 10 nucleotide forward primer (i.e., Ct_F10:5′-GCTTATGGAG-3′ (SEQ ID NO:3)) and a 10 nucleotide reverse primer(i.e., Ct_R10: 5′-ATACCGCTTA-3′ (SEQ ID NO:2)). The assay was designedto generate a 21 base DNA product, which included a one base spacer. Thespacer is a nucleotide between the 3′ ends of the primers, and thisnucleotide is not present in either of the primer sequences. The primers(i.e., 750 nM forward primer and 200 nM reverse primer) were mixed witheither TE as a NTC or different amounts of chlamydia genomic DNA (i.e.,20 copies, 200 copies, 1,000 copies, 2,000 copies) and placed inseparate reaction wells from the master mix solutions. In certaininstances, dH₂O was used as a NTC. The assay components were incubatedat 65° C. for 2 minutes, then combined to initiate the isothermalreaction. The reactions were carried out for 10 minutes at 65° C., andthen inactivated by placing on ice and adding EGTA. A molecular beacon(i.e., Ct FP MB5.18: Fam-CTGGCTACCGCTTAACTCCATAAGCCAG-3BHQ1 (SEQ IDNO:4)) containing a 20-base sequence complementary to the 21-basespecific forward product was then added to each reaction well. Thereaction products were detected by endpoint fluorescence readouts of themolecular beacon. As shown in FIG. 3, the isothermal reaction canamplify 20 copies of chlamydia genomic DNA to detectable levels in 10minutes at 65° C. using endpoint detection of a molecular beacon.

Chlamydia Genomic DNA Real-Time Detection by Molecular Beacon

Another approach for real-time detection of chlamydia genomic DNA is touse molecular beacons for detection. Under this approach, a 10nucleotide primer assay under asymmetric amplification conditions wasused for real-time molecular beacon detection with dH₂O or TE used as ano target control (NTC). Master mixes were prepared using 20 mM Tris-HClpH 8.8 at 25° C., 10 mM (NH₄)₂SO₄, 10 mM KCl, 4 mM MgSO₄, 0.1% Triton®X-100, 50 nM fMB2 3PS (molecular beacon), 0.25 mM dNTP, and 1unit/reaction of modified 9 Degrees North (9° Nm™) DNA polymerase (NewEngland BioLabs, Ipswich, Mass.). The master mix included a molecularbeacon (i.e., Ct_3PSMB.2: Fam-ccgcgagccttATACCGCTTAACTCg*c*g*g-IBFQ (SEQID NO:7)) which contained a 14-base sequence complementary to a portionof the forward product (14-base sequence is shown in upper caselettering). Nucleotides marked with * are phosphorothioate modified DNAbases. A primer set targeting a specific sequence within the 7,500 basepair C. trachomatis cryptic plasmid DNA was used, which included a 10nucleotide forward primer (i.e., Ct_F10+2: 5′-AGGCTTATGG-3′ (SEQ IDNO:5)) and a 10 nucleotide reverse primer (i.e., Ct_R10-2:5′-TTATACCGCT-3′ (SEQ ID NO:6)). The assay was designed to generate a 25base DNA product, which included a 5 base spacer. The spacer includes 5nucleotides between the 3′ ends of each primer, and these 5 nucleotidesare not present in either of the primer sequences. The primers (i.e.,750 nM forward primer and 200 nM reverse primer) were combined witheither TE as a NTC or 20,000 copies of chlamydia genomic DNA in reactionwells. In certain instances, dH₂O was used as a NTC. All components wereincubated at 65° C. for 2 minutes, then combined to initiate theisothermal reaction carried out at 65° C. The reaction products weredetected at various time points by real-time fluorescence readouts ofthe molecular beacon, as shown in FIG. 4.

Example 2: Examples of Sequences

Provided hereafter are non-limiting examples of certain nucleotide andamino acid sequences.

TABLE 1 Examples of sequences SEQ ID NO Name Type Sequence 1 Ct_F11 NAGGCTTATGGAG 2 Ct_R10 NA ATACCGCTTA 3 Ct_F10 NA GCTTATGGAG 4 Ct FP NACTGGCTACCGCTTAACTCCATAAGC MB5.18 CAG 5 Ct F10 +2 NA AGGCTTATGG 6Ct R10 −2 NA TTATACCGCT 7 Ct_3PSM NA ccgcgagccttATACCGCTTAACTC B.2g*c*g*g 8 9°N AA MILDTDYITENGKPVIRVFKKENGE FKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKF LGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKG LIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVITWKK IDLPYVDVVSTEKEMIKRFLRVVREKDPDVLITYNGDNFDFAYLKKRCEE LGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTL EAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGRE FFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDER ELARRRGGYAGGYVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNR EGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLE KKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVI RELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLL ELEYEGFYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQAR VLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKA TGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKH RYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK 9 9°Nm™ AA MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIED VKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRI RAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIDTLYHEGE EFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKRFLRVVRE KDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAV EVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWE SGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTG NLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGGYVKEPERGLWDNI VYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIP SLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRAIKILANSFYGYYGYA KARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPG ADAETVKKKAKEFLKYINPKLPGLLELEYEGFYVRGFFVTKKKYAVIDEE GKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKL SKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVIS YIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGY RKEDLRYQKTKQVGLGAWLKVKGKK *denotesphosphorothioate modified DNA bases

Example 3: Examples of Embodiments

The examples set forth below illustrate certain embodiments and do notlimit the technology.

A1. A method for amplifying nucleic acid, comprising:

-   -   contacting non-denatured sample nucleic acid under isothermal        amplification conditions with components comprising    -   a) at least one oligonucleotide, which at least one        oligonucleotide comprises a polynucleotide complementary to a        target sequence in the sample nucleic acid, and    -   b) at least one component providing hyperthermophile polymerase        activity, thereby generating a nucleic acid amplification        product.

A1.1 A method for amplifying nucleic acid, comprising:

-   -   contacting non-denatured sample nucleic acid under isothermal        amplification conditions with    -   a) non-enzymatic components comprising at least one        oligonucleotide, which at least one oligonucleotide comprises a        polynucleotide complementary to a target sequence in the sample        nucleic acid, and    -   b) an enzymatic component consisting of a hyperthermophile        polymerase or a polymerase comprising an amino acid sequence        that is at least about 90% identical to a hyperthermophile        polymerase,    -   thereby generating a nucleic acid amplification product.

A1.2 A method for amplifying nucleic acid, comprising:

-   -   contacting non-denatured sample nucleic acid under isothermal        amplification conditions with    -   a) non-enzymatic components comprising at least one        oligonucleotide, which at least one oligonucleotide comprises a        polynucleotide complementary to a target sequence in the sample        nucleic acid, and    -   b) enzymatic activity consisting of i) hyperthermophile        polymerase activity and, optionally, ii) reverse transcriptase        activity,    -   thereby generating a nucleic acid amplification product.

A1.3 The method of embodiment A1.2, wherein the enzymatic activityconsists of i) hyperthermophile polymerase activity, and ii) reversetranscriptase activity.

A2. The method of any one of embodiments A1 to A1.3, wherein the methoddoes not comprise denaturing the sample nucleic acid prior to or duringamplification.

A3. The method of any one of embodiments A1 to A2, wherein the samplenucleic acid is not contacted with an endonuclease prior to or duringamplification.

A4. The method of any one of embodiments A1 to A3, wherein the samplenucleic acid is not contacted with an unwinding agent prior to or duringamplification.

A5. The method of any one of embodiments A1 to A4, wherein the samplenucleic acid is not contacted with a helicase prior to or duringamplification.

A5.1 The method of any one of embodiments A1 to A5, wherein the samplenucleic acid is not contacted with a recombinase prior to or duringamplification.

A5.2 The method of any one of embodiments A1 to A5.1, wherein the samplenucleic acid is not contacted with a single-stranded DNA binding proteinprior to or during amplification.

A6. The method of any one of embodiments A1 to A5.2, wherein the samplenucleic acid is unmodified prior to amplification.

A7. The method of embodiment A6, wherein the unmodified sample nucleicacid is from disrupted cells.

A8. The method of any one of embodiments A1 to A7, wherein the samplenucleic acid comprises DNA.

A9. The method of embodiment A8, wherein the sample nucleic acidcomprises genomic DNA.

A10. The method of any one of embodiments A1 to A7, wherein the samplenucleic acid comprises RNA.

A11. The method of embodiment A10, wherein the sample nucleic acidcomprises viral RNA.

A12. The method of embodiment A10, wherein the sample nucleic acidcomprises bacterial RNA.

A13. The method of any one of embodiments A1 to A12, wherein the samplenucleic acid comprises single-stranded nucleic acid.

A14. The method of any one of embodiments A1 to A12, wherein the samplenucleic acid comprises double-stranded nucleic acid, whichdouble-stranded nucleic acid comprises a first strand and a secondstrand.

A15. The method of any one of embodiments A1 to A14, wherein the atleast one oligonucleotide comprises a first oligonucleotide and a secondoligonucleotide.

A16. The method of any one of embodiments A1 to A14, wherein the atleast one oligonucleotide consists of a first oligonucleotide and asecond oligonucleotide.

A16.1 The method of embodiment A15 or A16, wherein the firstoligonucleotide and the second oligonucleotide each comprise 8 to 16bases.

A17. The method of embodiment A15, A16 or A16.1, wherein the firstoligonucleotide comprises a first polynucleotide complementary to atarget sequence in the first strand of the sample nucleic acid, and thesecond oligonucleotide comprises a second polynucleotide complementaryto a target sequence in the second strand of the sample nucleic acid.

A18. The method of embodiment A15, A16 or A16.1, wherein the firstoligonucleotide comprises a first polynucleotide continuouslycomplementary to a target sequence in the first strand of the samplenucleic acid, and the second oligonucleotide comprises a secondpolynucleotide continuously complementary to a target sequence in thesecond strand of the sample nucleic acid.

A19. The method of embodiment A15, A16 or A16.1, wherein the firstoligonucleotide consists of a first polynucleotide continuouslycomplementary to a target sequence in the first strand of the samplenucleic acid, and the second oligonucleotide consists of a secondpolynucleotide continuously complementary to a target sequence in thesecond strand of the sample nucleic acid.

A20. The method of any one of embodiments A1 to A19, wherein samplenucleic acid is obtained from a subject prior to amplification.

A21. The method of any one of embodiments A1 to A20, wherein unpurifiedsample nucleic acid is amplified.

A22. The method of any one of embodiments A1 to A20, wherein purifiedsample nucleic acid is amplified.

A23. The method of any one of embodiments A1 to A20, further comprisingpurifying sample nucleic acid prior to amplification.

A24. The method of any one of embodiments A1 to A23, wherein thehyperthermophile polymerase activity is provided by a hyperthermophilepolymerase or functional fragment thereof.

A25. The method of any one of embodiments A1 to A23, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence that is at least about 90% identicalto a hyperthermophile polymerase or functional fragment thereof.

A26. The method of any one of embodiments A1 to A23, wherein thehyperthermophile polymerase activity is provided by an Archaeahyperthermophile polymerase or functional fragment thereof.

A27. The method of any one of embodiments A1 to A26, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence of SEQ ID NO:8 or functional fragmentthereof.

A28. The method of any one of embodiments A1 to A26, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence that is at least about 90% identicalto the amino acid sequence of SEQ ID NO:8 or functional fragmentthereof.

A28.1 The method of any one of embodiments A1 to A28, wherein thehyperthermophile polymerase activity is provided by a polymerase havinglow exonuclease activity.

A28.2 The method of any one of embodiments A1 to A28, wherein thehyperthermophile polymerase activity is provided by a polymerase havingno exonuclease activity.

A29. The method of any one of embodiments A1 to A28.2, wherein theamplification is performed at a constant temperature of about 55 degreesCelsius to about 75 degrees Celsius.

A30. The method of any one of embodiments A1 to A28.2, wherein theamplification is performed at a constant temperature of about 55 degreesCelsius to about 65 degrees Celsius.

A31. The method of any one of embodiments A1 to A28.2, wherein theamplification is performed at a constant temperature of about 65 degreesCelsius.

A32. The method of any one of embodiments A1 to A28.2, wherein theamplification is performed at a constant temperature of about 60 degreesCelsius.

A33. The method of any one of embodiments A1 to A32, wherein the nucleicacid amplification product is detectable in 10 minutes or less.

A34. The method of any one of embodiments A1 to A33, wherein the nucleicacid amplification product comprises a polynucleotide that iscontinuously complementary to or substantially identical to a targetsequence in the sample nucleic acid.

A35. The method of any one of embodiments A1 to A33, wherein the nucleicacid amplification product consists of a polynucleotide that iscontinuously complementary to or substantially identical to a targetsequence in the sample nucleic acid.

A36. The method of any one of embodiments A1 to A35, wherein the nucleicacid amplification product is about 20 to 40 bases long.

A37. The method of any one of embodiments A16 to A36, wherein thenucleic acid amplification product comprises i) a first nucleotidesequence that is continuously complementary to or substantiallyidentical to the first polynucleotide of the first oligonucleotide, ii)a second nucleotide sequence that is continuously complementary to orsubstantially identical to the second polynucleotide of the secondoligonucleotide, and iii) a spacer sequence, wherein the spacer sequenceis flanked by the first nucleotide sequence and the second nucleotidesequence.

A38. The method of any one of embodiments A16 to A36, wherein thenucleic acid amplification product consists of i) a first nucleotidesequence that is continuously complementary to or substantiallyidentical to the first polynucleotide of the first oligonucleotide, ii)a second nucleotide sequence that is continuously complementary to orsubstantially identical to the second polynucleotide of the secondoligonucleotide, and iii) a spacer sequence, wherein the spacer sequenceis flanked by the first nucleotide sequence and the second nucleotidesequence.

A39. The method of embodiment A37 or A38, wherein the spacer sequencecomprises 1 to 10 bases.

A40. The method of embodiment A37 or A38, wherein the spacer sequencecomprises 1 to 5 bases.

A41. The method of any one of embodiments A37 to A40, wherein the spacersequence is not complementary to or identical to the firstpolynucleotide of the first oligonucleotide and is not complementary toor identical to the second polynucleotide of the second oligonucleotide.

A42. The method of any one of embodiments A37 to A41, wherein the spacersequence is continuously complementary to or substantially identical toa portion of a target sequence in the sample nucleic acid.

A43. The method of any one of embodiments A1 to A42, further comprisingdetecting the nucleic acid amplification product.

A44. The method of embodiment A43, wherein detecting the nucleic acidamplification product is performed in 10 minutes or less from the timethe sample nucleic acid is contacted with the component providing thehyperthermophile polymerase activity and the at least oneoligonucleotide.

A45. The method of embodiment A43 or A44, wherein detecting the nucleicacid amplification product comprises use of a real-time detectionmethod.

A46. The method of embodiment A43, A44 or A45, wherein detecting thenucleic acid amplification product comprises detection of a fluorescentsignal.

A47. The method of embodiment A46, wherein the fluorescent signal isfrom a molecular beacon.

A48. The method of any one of embodiments A1 to A47, further comprisingcontacting the nucleic acid amplification product with a signalgenerating oligonucleotide that comprises i) a polynucleotidecomplementary to a sequence in the amplification product, and ii) afluorophore and a quencher.

A49. The method of any one of embodiments A1 to A47, wherein one or moreof the at least one oligonucleotide comprise a polynucleotide notcomplementary to a sequence in the sample nucleic acid that hybridizesto a signal generating oligonucleotide, and wherein the method furthercomprises contacting the amplification product with the signalgenerating oligonucleotide that comprises a fluorophore and a quencher.

A50. The method of any one of embodiments A1 to A49, wherein the methodis performed in a single reaction volume.

A51. The method of any one of embodiments A1 to A50, wherein the methodis performed in a single reaction vessel.

A52. The method of any one of embodiments A1 to A51, comprisingmultiplex amplification.

B1. A method for processing nucleic acid, comprising:

-   -   amplifying nucleic acid, wherein the amplifying consists        essentially of contacting non-denatured sample nucleic acid        under isothermal amplification conditions with    -   a) at least one oligonucleotide, which at least one        oligonucleotide comprises a polynucleotide complementary to a        target sequence in the sample nucleic acid, and    -   b) at least one component providing hyperthermophile polymerase        activity, thereby generating a nucleic acid amplification        product.

B2. A method for processing nucleic acid, comprising:

-   -   amplifying nucleic acid, wherein the amplifying consists        essentially of contacting non-denatured sample nucleic acid        under isothermal amplification conditions with    -   a) non-enzymatic components comprising at least one        oligonucleotide, which at least one oligonucleotide comprises a        polynucleotide complementary to a target sequence in the sample        nucleic acid, and    -   b) an enzymatic component consisting of a hyperthermophile        polymerase or a polymerase comprising an amino acid sequence        that is at least about 90% identical to a hyperthermophile        polymerase,    -   thereby generating a nucleic acid amplification product.

B3. A method for processing nucleic acid, comprising:

-   -   amplifying nucleic acid, wherein the amplifying consists        essentially of contacting non-denatured sample nucleic acid        under isothermal amplification conditions with    -   a) non-enzymatic components comprising at least one        oligonucleotide, which at least one oligonucleotide comprises a        polynucleotide complementary to a target sequence in the sample        nucleic acid, and    -   b) enzymatic activity consisting of i) hyperthermophile        polymerase activity and, optionally, ii) reverse transcriptase        activity,    -   thereby generating a nucleic acid amplification product.

B4. The method of embodiment B3, wherein the enzymatic activity consistsof i) hyperthermophile polymerase activity, and ii) reversetranscriptase activity.

B5. A method for processing nucleic acid, comprising:

-   -   amplifying nucleic acid, wherein the amplifying consists of        contacting non-denatured sample nucleic acid under isothermal        amplification conditions with    -   a) at least one oligonucleotide, which at least one        oligonucleotide comprises a polynucleotide complementary to a        target sequence in the sample nucleic acid, and    -   b) at least one component providing hyperthermophile polymerase        activity, thereby generating a nucleic acid amplification        product.

B6. A method for processing nucleic acid, comprising:

-   -   amplifying nucleic acid, wherein the amplifying consists of        contacting non-denatured sample nucleic acid under isothermal        amplification conditions with    -   a) non-enzymatic components comprising at least one        oligonucleotide, which at least one oligonucleotide comprises a        polynucleotide complementary to a target sequence in the sample        nucleic acid, and    -   b) an enzymatic component consisting of a hyperthermophile        polymerase or a polymerase comprising an amino acid sequence        that is at least about 90% identical to a hyperthermophile        polymerase,    -   thereby generating a nucleic acid amplification product.

B7. A method for processing nucleic acid, comprising:

-   -   amplifying nucleic acid, wherein the amplifying consists of        contacting non-denatured sample nucleic acid under isothermal        amplification conditions with    -   a) non-enzymatic components comprising at least one        oligonucleotide, which at least one oligonucleotide comprises a        polynucleotide complementary to a target sequence in the sample        nucleic acid, and    -   b) enzymatic activity consisting of i) hyperthermophile        polymerase activity and, optionally, ii) reverse transcriptase        activity,    -   thereby generating a nucleic acid amplification product.

B8. The method of embodiment B7, wherein the enzymatic activity consistsof i) hyperthermophile polymerase activity, and ii) reversetranscriptase activity.

B9. The method of any one of embodiments B1 to B8, wherein the samplenucleic acid is unmodified prior to amplification.

B10. The method of embodiment B9, wherein the unmodified sample nucleicacid is from disrupted cells.

B11. The method of any one of embodiments B1 to B10, wherein the samplenucleic acid comprises DNA.

B12. The method of embodiment B11, wherein the sample nucleic acidcomprises genomic DNA.

B13. The method of any one of embodiments B1 to B10, wherein the samplenucleic acid comprises RNA.

B14. The method of embodiment B13, wherein the sample nucleic acidcomprises viral RNA.

B15. The method of embodiment B13, wherein the sample nucleic acidcomprises bacterial RNA.

B16. The method of any one of embodiments B1 to B15, wherein the samplenucleic acid comprises single-stranded nucleic acid.

B17. The method of any one of embodiments B1 to B15, wherein the samplenucleic acid comprises double-stranded nucleic acid, whichdouble-stranded nucleic acid comprises a first strand and a secondstrand.

B18. The method of any one of embodiments B1 to B17, wherein the atleast one oligonucleotide comprises a first oligonucleotide and a secondoligonucleotide.

B19. The method of any one of embodiments B1 to B17, wherein the atleast one oligonucleotide consists of a first oligonucleotide and asecond oligonucleotide.

B19.1 The method of embodiment B18 or B19, wherein the firstoligonucleotide and the second oligonucleotide each comprise 8 to 16bases.

B20. The method of embodiment B18, B19 or B19.1, wherein the firstoligonucleotide comprises a first polynucleotide complementary to atarget sequence in the first strand of the sample nucleic acid, and thesecond oligonucleotide comprises a second polynucleotide complementaryto a target sequence in the second strand of the sample nucleic acid.

B21. The method of embodiment B18, B19 or B19.1, wherein the firstoligonucleotide comprises a first polynucleotide continuouslycomplementary to a target sequence in the first strand of the samplenucleic acid, and the second oligonucleotide comprises a secondpolynucleotide continuously complementary to a target sequence in thesecond strand of the sample nucleic acid.

B22. The method of embodiment B18, B19 or B19.1, wherein the firstoligonucleotide consists of a first polynucleotide continuouslycomplementary to a target sequence in the first strand of the samplenucleic acid, and the second oligonucleotide consists of a secondpolynucleotide continuously complementary to a target sequence in thesecond strand of the sample nucleic acid.

B23. The method of any one of embodiments B1 to B22, wherein samplenucleic acid is obtained from a subject prior to amplification.

B24. The method of any one of embodiments B1 to B22, wherein unpurifiedsample nucleic acid is amplified.

B25. The method of any one of embodiments B1 to B22, wherein purifiedsample nucleic acid is amplified.

B26. The method of any one of embodiments B1 to B22, further comprisingpurifying sample nucleic acid prior to amplification.

B27. The method of any one of embodiments B1 to B26, wherein thehyperthermophile polymerase activity is provided by a hyperthermophilepolymerase or functional fragment thereof.

B28. The method of any one of embodiments B1 to B26, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence that is at least about 90% identicalto a hyperthermophile polymerase or functional fragment thereof.

B29. The method of any one of embodiments B1 to B26, wherein thehyperthermophile polymerase activity is provided by an Archaeahyperthermophile polymerase or functional fragment thereof.

B30. The method of any one of embodiments B1 to B29, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence of SEQ ID NO:8 or functional fragmentthereof.

B31. The method of any one of embodiments B1 to B29, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence that is at least about 90% identicalto the amino acid sequence of SEQ ID NO:8 or functional fragmentthereof.

B31.1 The method of any one of embodiments B1 to B31, wherein thehyperthermophile polymerase activity is provided by a polymerase havinglow exonuclease activity.

B32.2 The method of any one of embodiments B1 to B31, wherein thehyperthermophile polymerase activity is provided by a polymerase havingno exonuclease activity.

B32. The method of any one of embodiments B1 to B31.2, wherein theamplification is performed at a constant temperature of about 55 degreesCelsius to about 75 degrees Celsius.

B33. The method of any one of embodiments B1 to B31.2, wherein theamplification is performed at a constant temperature of about 55 degreesCelsius to about 65 degrees Celsius.

B34. The method of any one of embodiments B1 to B31.2, wherein theamplification is performed at a constant temperature of about 65 degreesCelsius.

B35. The method of any one of embodiments B1 to B31.2, wherein theamplification is performed at a constant temperature of about 60 degreesCelsius.

B36. The method of any one of embodiments B1 to B35, wherein the nucleicacid amplification product is detectable in 10 minutes or less.

B37. The method of any one of embodiments B1 to B36, wherein the nucleicacid amplification product comprises a polynucleotide that iscontinuously complementary to or substantially identical to a targetsequence in the sample nucleic acid.

B38. The method of any one of embodiments B1 to B36, wherein the nucleicacid amplification product consists of a polynucleotide that iscontinuously complementary to or substantially identical to a targetsequence in the sample nucleic acid.

B39. The method of any one of embodiments B1 to B38, wherein the nucleicacid amplification product is about 20 to 40 bases long.

B40. The method of any one of embodiments B20 to B39, wherein thenucleic acid amplification product comprises i) a first nucleotidesequence that is continuously complementary to or substantiallyidentical to the first polynucleotide of the first oligonucleotide, ii)a second nucleotide sequence that is continuously complementary to orsubstantially identical to the second polynucleotide of the secondoligonucleotide, and iii) a spacer sequence, wherein the spacer sequenceis flanked by the first nucleotide sequence and the second nucleotidesequence.

B41. The method of any one of embodiments B20 to B39, wherein thenucleic acid amplification product consists of i) a first nucleotidesequence that is continuously complementary to or substantiallyidentical to the first polynucleotide of the first oligonucleotide, ii)a second nucleotide sequence that is continuously complementary to orsubstantially identical to the second polynucleotide of the secondoligonucleotide, and iii) a spacer sequence, wherein the spacer sequenceis flanked by the first nucleotide sequence and the second nucleotidesequence.

B42. The method of embodiment B40 or B41, wherein the spacer sequencecomprises 1 to 10 bases.

B43. The method of embodiment B40 or B41, wherein the spacer sequencecomprises 1 to 5 bases.

B44. The method of any one of embodiments B40 to B43, wherein the spacersequence is not complementary to or identical to the firstpolynucleotide of the first oligonucleotide and is not complementary toor identical to the second polynucleotide of the second oligonucleotide.

B45. The method of any one of embodiments B40 to B44, wherein the spacersequence is continuously complementary to or substantially identical toa portion of a target sequence in the sample nucleic acid.

B46. The method of any one of embodiments B1 to B45, further comprisingdetecting the nucleic acid amplification product.

B47. The method of embodiment B46, wherein detecting the nucleic acidamplification product is performed in 10 minutes or less from the timethe sample nucleic acid is contacted with the component providing thehyperthermophile polymerase activity and the at least oneoligonucleotide.

B48. The method of embodiment B46 or B47, wherein detecting the nucleicacid amplification product comprises use of a real-time detectionmethod.

B49. The method of embodiment B46, B47 or B48, wherein detecting thenucleic acid amplification product comprises detection of a fluorescentsignal.

B50. The method of embodiment B49, wherein the fluorescent signal isfrom a molecular beacon.

B51. The method of any one of embodiments B1 to B50, further comprisingcontacting the nucleic acid amplification product with a signalgenerating oligonucleotide that comprises i) a polynucleotidecomplementary to a sequence in the amplification product, and ii) afluorophore and a quencher.

B52. The method of any one of embodiments B1 to B50, wherein one or moreof the at least one oligonucleotide comprise a polynucleotide notcomplementary to a sequence in the sample nucleic acid that hybridizesto a signal generating oligonucleotide, and wherein the method furthercomprises contacting the amplification product with the signalgenerating oligonucleotide that comprises a fluorophore and a quencher.

B53. The method of any one of embodiments B1 to B52, wherein the methodis performed in a single reaction volume.

B54. The method of any one of embodiments B1 to B53, wherein the methodis performed in a single reaction vessel.

B55. The method of any one of embodiments B1 to B54, comprisingmultiplex amplification.

C1. A method for determining the presence, absence or amount of a targetsequence in sample nucleic acid, comprising:

-   -   a) amplifying a target sequence in the sample nucleic acid,        wherein:        -   the target sequence comprises a first strand and a second            strand, the first strand and second strand are complementary            to each other, and the amplifying comprises contacting            non-denatured sample nucleic acid under helicase-free            isothermal amplification conditions with:        -   i) a first oligonucleotide and a second oligonucleotide,            wherein the first oligonucleotide comprises a first            polynucleotide continuously complementary to a sequence in            the first strand, and the second oligonucleotide comprises a            second polynucleotide continuously complementary to a            sequence in the second strand; and        -   ii) at least one component providing a hyperthermophile            polymerase activity, thereby generating a nucleic acid            amplification product, wherein the nucleic acid            amplification product comprises 1) a first nucleotide            sequence that is continuously complementary to or            substantially identical to the first polynucleotide of the            first oligonucleotide, 2) a second nucleotide sequence that            is continuously complementary to or substantially identical            to the second polynucleotide of the second oligonucleotide,            and 3) a spacer sequence comprising 1 to 10 bases, and    -   the spacer sequence is flanked by the first nucleotide sequence        and the second nucleotide sequence; and    -   b) detecting the nucleic acid amplification product, wherein        detecting the nucleic acid amplification product comprises use        of a real-time detection method and is performed in 10 minutes        or less from the time the sample nucleic acid is contacted with        (a)(i) and (a)(ii), whereby the presence, absence or amount of a        target sequence in sample nucleic acid is determined.

C1.1 The method of embodiment C1, wherein the first oligonucleotideconsists essentially of a first polynucleotide continuouslycomplementary to a sequence in the first strand, and the secondoligonucleotide consists essentially of a second polynucleotidecontinuously complementary to a sequence in the second strand; and/orthe nucleic acid amplification product consists essentially of 1) afirst nucleotide sequence that is continuously complementary to orsubstantially identical to the first polynucleotide of the firstoligonucleotide, 2) a second nucleotide sequence that is continuouslycomplementary to or substantially identical to the second polynucleotideof the second oligonucleotide, and 3) a spacer sequence comprising 1 to10 bases.

C1.2 The method of embodiment C1, wherein the first oligonucleotideconsists of a first polynucleotide continuously complementary to asequence in the first strand, and the second oligonucleotide consists ofa second polynucleotide continuously complementary to a sequence in thesecond strand; and/or the nucleic acid amplification product consistsof 1) a first nucleotide sequence that is continuously complementary toor substantially identical to the first polynucleotide of the firstoligonucleotide, 2) a second nucleotide sequence that is continuouslycomplementary to or substantially identical to the second polynucleotideof the second oligonucleotide, and 3) a spacer sequence comprising 1 to10 bases.

C1.3 A method for determining the presence, absence or amount of atarget sequence in sample nucleic acid, comprising:

-   -   a) amplifying a target sequence in the sample nucleic acid,        wherein:    -   the target sequence comprises a first strand and a second        strand, the first strand and second strand are complementary to        each other, and the amplifying comprises contacting        non-denatured sample nucleic acid under helicase-free isothermal        amplification conditions with:        -   i) a first oligonucleotide and a second oligonucleotide,            wherein the first oligonucleotide consists of a first            polynucleotide continuously complementary to a sequence in            the first strand, and the second oligonucleotide consists of            a second polynucleotide continuously complementary to a            sequence in the second strand; and        -   ii) at least one component providing a hyperthermophile            polymerase activity, thereby generating a nucleic acid            amplification product, wherein the nucleic acid            amplification product consists of 1) a first nucleotide            sequence that is continuously complementary to or            substantially identical to the first polynucleotide of the            first oligonucleotide, 2) a second nucleotide sequence that            is continuously complementary to or substantially identical            to the second polynucleotide of the second oligonucleotide,            and 3) a spacer sequence comprising 1 to 10 bases, and    -   the spacer sequence is flanked by the first nucleotide sequence        and the second nucleotide sequence; and    -   b) detecting the nucleic acid amplification product, wherein        detecting the nucleic acid amplification product comprises use        of a real-time detection method and is performed in 10 minutes        or less from the time the sample nucleic acid is contacted with        (a)(i) and (a)(ii), whereby the presence, absence or amount of a        target sequence in sample nucleic acid is determined.

C1.4 The method of any one of embodiments C1 to C1.3, wherein theamplifying comprises contacting non-denatured sample nucleic acid underhelicase-free and recombinase-free isothermal amplification conditions.

C2. The method of any one of embodiments C1 to C1.4, wherein the atleast one component providing a hyperthermophile polymerase activitycomprises a hyperthermophile polymerase or functional fragment thereof,or a polymerase comprising an amino acid sequence that is at least about90% identical to a hyperthermophile polymerase or functional fragmentthereof.

C3. The method of any one of embodiments C1 to C1.4, wherein the atleast one component providing a hyperthermophile polymerase activityconsists of a hyperthermophile polymerase or functional fragmentthereof, or a polymerase comprising an amino acid sequence that is atleast about 90% identical to a hyperthermophile polymerase or functionalfragment thereof.

C4. The method of any one of embodiments C1 to C3, wherein thehyperthermophile polymerase activity is provided by an Archaeahyperthermophile polymerase or functional fragment thereof.

C5. The method of any one of embodiments C1 to C4, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence of SEQ ID NO:8 or functional fragmentthereof.

C6. The method of any one of embodiments C1 to C4, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence that is at least about 90% identicalto the amino acid sequence of SEQ ID NO:8 or functional fragmentthereof.

C6.1 The method of any one of embodiments C1 to C6, wherein thehyperthermophile polymerase activity is provided by a polymerase havinglow exonuclease activity.

C6.2 The method of any one of embodiments C1 to C6, wherein thehyperthermophile polymerase activity is provided by a polymerase havingno exonuclease activity.

C7. The method of any one of embodiments C1 to C6.2, wherein part(a)(ii) further comprises at least one component providing a reversetranscriptase activity.

C8. The method of any one of embodiments C1 to C6.2, wherein the atleast one component providing hyperthermophile polymerase activityfurther provides a reverse transcriptase activity.

C8.1 The method of any one of embodiments C1 to C8, wherein the firstoligonucleotide and the second oligonucleotide each comprise 8 to 16bases.

C9. The method of any one of embodiments C1 to C8.1, wherein the methoddoes not comprise denaturing the sample nucleic acid prior to or duringamplification.

C10. The method of any one of embodiments C1 to C9, wherein the samplenucleic acid is not contacted with an endonuclease prior to, during, orfollowing amplification.

C10.1 The method of any one of embodiments C1 to C10, wherein the samplenucleic acid is not contacted with a recombinase prior to or duringamplification.

C10.2 The method of any one of embodiments C1 to C10.1, wherein thesample nucleic acid is not contacted with a single-stranded DNA bindingprotein prior to or during amplification.

C11. The method of any one of embodiments C1 to C10.2, wherein thesample nucleic acid is unmodified prior to amplification.

C12. The method of embodiment C11, wherein the unmodified sample nucleicacid is from disrupted cells.

C13. The method of any one of embodiments C1 to C12, wherein the samplenucleic acid comprises DNA.

C14. The method of embodiment C13, wherein the sample nucleic acidcomprises genomic DNA.

C15. The method of any one of embodiments C1 to C12, wherein the samplenucleic acid comprises RNA.

C16. The method of embodiment C15, wherein the sample nucleic acidcomprises viral RNA.

C17. The method of embodiment C15, wherein the sample nucleic acidcomprises bacterial RNA.

C18. The method of any one of embodiments C1 to C17, wherein the samplenucleic acid comprises single-stranded nucleic acid.

C19. The method of any one of embodiments C1 to C17, wherein the samplenucleic acid comprises double-stranded nucleic acid.

C20. The method of any one of embodiments C1 to C19, wherein samplenucleic acid is obtained from a subject prior to amplification.

C20.1 The method of any one of embodiments C1 to C20, wherein unpurifiedsample nucleic acid is amplified.

C20.2 The method of any one of embodiments C1 to C20, wherein purifiedsample nucleic acid is amplified.

C21. The method of any one of embodiments C1 to C20.2, furthercomprising purifying sample nucleic acid prior to amplification.

C22. The method of any one of embodiments C1 to C21, wherein theamplification is performed at a constant temperature of about 55 degreesCelsius to about 75 degrees Celsius.

C23. The method of any one of embodiments C1 to C21, wherein theamplification is performed at a constant temperature of about 55 degreesCelsius to about 65 degrees Celsius.

C24. The method of any one of embodiments C1 to C21, wherein theamplification is performed at a constant temperature of about 65 degreesCelsius.

C25. The method of any one of embodiments C1 to C21, wherein theamplification is performed at a constant temperature of about 60 degreesCelsius.

C26. The method of any one of embodiments C1 to C25, wherein the nucleicacid amplification product is about 20 to 40 bases long.

C27. The method of any one of embodiments C1 to C26, wherein the spacersequence comprises 1 to 5 bases.

C28. The method of any one of embodiments C1 to C27, wherein the spacersequence is not complementary to or identical to the firstpolynucleotide of the first oligonucleotide and is not complementary toor identical to the second polynucleotide of the second oligonucleotide.

C29. The method of any one of embodiments C1 to C28, wherein the spacersequence is continuously complementary to or substantially identical toa portion of a target sequence in the sample nucleic acid.

C30. The method of any one of embodiments C1 to C28, wherein detectingthe nucleic acid amplification product comprises detection of afluorescent signal.

C31. The method embodiment C30, wherein the fluorescent signal is from amolecular beacon.

C32. The method of any one of embodiments C1 to C31, further comprisingcontacting the nucleic acid amplification product with a signalgenerating oligonucleotide that comprises i) a polynucleotidecomplementary to a sequence in the amplification product, and ii) afluorophore and a quencher.

C33. The method of any one of embodiments C1 to C32, wherein the methodis performed in a single reaction volume.

C34. The method of any one of embodiments C1 to C33, wherein the methodis performed in a single reaction vessel.

C35. The method of any one of embodiments C1 to C34, comprisingmultiplex amplification.

D1. A kit for determining the presence, absence or amount of a targetsequence in sample nucleic acid comprising:

-   -   a) components for amplifying a target sequence in the sample        nucleic acid under helicase-free isothermal amplification        conditions, which components comprise:        -   i) a first oligonucleotide and a second oligonucleotide,            wherein the first oligonucleotide comprises a first            polynucleotide continuously complementary to a sequence in a            first strand of the target sequence, and the second            oligonucleotide comprises a second polynucleotide            continuously complementary to a sequence in a second strand            of the target sequence, which first strand and second strand            of the target sequence are complementary to each other; and        -   ii) at least one component providing a hyperthermophile            polymerase activity; and    -   b) at least one component providing real-time detection activity        for a nucleic acid amplification product.

D1.1 The kit of embodiment D1, wherein the first oligonucleotideconsists essentially of a first polynucleotide continuouslycomplementary to a sequence in a first strand of the target sequence,and the second oligonucleotide consists essentially of a secondpolynucleotide continuously complementary to a sequence in a secondstrand of the target sequence.

D1.2 The kit of embodiment D1, wherein the first oligonucleotideconsists of a first polynucleotide continuously complementary to asequence in a first strand of the target sequence, and the secondoligonucleotide consists of a second polynucleotide continuouslycomplementary to a sequence in a second strand of the target sequence.

D1.3 A kit for determining the presence, absence or amount of a targetsequence in sample nucleic acid comprising:

-   -   a) components for amplifying a target sequence in the sample        nucleic acid under helicase-free isothermal amplification        conditions, which components comprise:        -   i) a first oligonucleotide and a second oligonucleotide,            wherein the first oligonucleotide consists of a first            polynucleotide continuously complementary to a sequence in a            first strand of the target sequence, and the second            oligonucleotide consists of a second polynucleotide            continuously complementary to a sequence in a second strand            of the target sequence, which first strand and second strand            of the target sequence are complementary to each other; and        -   ii) at least one component providing a hyperthermophile            polymerase activity; and    -   b) at least one component providing real-time detection activity        for a nucleic acid amplification product.

D1.4 The kit of any one of embodiments D1 to D1.3, wherein the samplenucleic acid is amplified under helicase-free and recombinase-freeisothermal amplification conditions.

D2. The kit of any one of embodiments D1 to D1.4, wherein the at leastone component providing a hyperthermophile polymerase activity comprisesa hyperthermophile polymerase or functional fragment thereof, or apolymerase comprising an amino acid sequence that is at least about 90%identical to a hyperthermophile polymerase or functional fragmentthereof.

D3. The kit of any one of embodiments D1 to D1.4, wherein the at leastone component providing a hyperthermophile polymerase activity consistsof a hyperthermophile polymerase or functional fragment thereof, or apolymerase comprising an amino acid sequence that is at least about 90%identical to a hyperthermophile polymerase or functional fragmentthereof.

D4. The kit of any one of embodiments D1 to D3, wherein thehyperthermophile polymerase activity is provided by an Archaeahyperthermophile polymerase or functional fragment thereof.

D5. The kit of any one of embodiments D1 to D4, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence of SEQ ID NO:8 or functional fragmentthereof.

D6. The kit of any one of embodiments D1 to D4, wherein thehyperthermophile polymerase activity is provided by a polymerasecomprising an amino acid sequence that is at least about 90% identicalto the amino acid sequence of SEQ ID NO:8 or functional fragmentthereof.

D7. The kit of any one of embodiments D1 to D6, wherein thehyperthermophile polymerase activity is provided by a polymerase havinglow exonuclease activity.

D8. The kit of any one of embodiments D1 to D6, wherein thehyperthermophile polymerase activity is provided by a polymerase havingno exonuclease activity.

D9. The kit of any one of embodiments D1 to D8, wherein part (a)(ii)further comprises at least one component providing a reversetranscriptase activity.

D10. The kit of any one of embodiments D1 to D8, wherein the at leastone component providing hyperthermophile polymerase activity furtherprovides a reverse transcriptase activity.

D11. The kit of any one of embodiments D1 to D10, wherein the firstoligonucleotide and the second oligonucleotide each comprise 8 to 16bases.

D12. The kit of any one of embodiments D1 to D11, wherein the real-timedetection activity is provided by a molecular beacon.

D13. The kit of any one of embodiments D1 to D12, further comprisinginstructions for carrying out a method for determining the presence,absence or amount of a target sequence in sample nucleic acid, themethod comprising:

-   -   a) amplifying a target sequence in the sample nucleic acid,        wherein:    -   the target sequence comprises a first strand and a second        strand,    -   the first strand and second strand are complementary to each        other,    -   and the amplifying comprises contacting non-denatured sample        nucleic acid under helicase-free isothermal amplification        conditions with:        -   i) a first oligonucleotide and a second oligonucleotide,            wherein the first oligonucleotide comprises a first            polynucleotide continuously complementary to a sequence in            the first strand, and the second oligonucleotide comprises a            second polynucleotide continuously complementary to a            sequence in the second strand; and        -   ii) at least one component providing a hyperthermophile            polymerase activity, thereby generating a nucleic acid            amplification product, wherein the nucleic acid            amplification product comprises 1) a first nucleotide            sequence that is continuously complementary to or            substantially identical to the first polynucleotide of the            first oligonucleotide, 2) a second nucleotide sequence that            is continuously complementary to or substantially identical            to the second polynucleotide of the second oligonucleotide,            and 3) a spacer sequence comprising 1 to 10 bases, and the            spacer sequence is flanked by the first nucleotide sequence            and the second nucleotide sequence; and    -   b) detecting the nucleic acid amplification product, wherein        detecting the nucleic acid amplification product comprises use        of a real-time detection method and is performed in 10 minutes        or less from the time the sample nucleic acid is contacted with        (a)(i) and (a)(ii), whereby the presence, absence or amount of a        target sequence in sample nucleic acid is determined. D13.1 The        kit of embodiment D13, wherein the first oligonucleotide        consists essentially of a first polynucleotide continuously        complementary to a sequence in the first strand, and the second        oligonucleotide consists essentially of a second polynucleotide        continuously complementary to a sequence in the second strand;        and/or the nucleic acid amplification product consists        essentially of 1) a first nucleotide sequence that is        continuously complementary to or substantially identical to the        first polynucleotide of the first oligonucleotide, 2) a second        nucleotide sequence that is continuously complementary to or        substantially identical to the second polynucleotide of the        second oligonucleotide, and 3) a spacer sequence comprising 1 to        10 bases.

D13.2 The kit of embodiment D13, wherein the first oligonucleotideconsists of a first polynucleotide continuously complementary to asequence in the first strand, and the second oligonucleotide consists ofa second polynucleotide continuously complementary to a sequence in thesecond strand; and/or the nucleic acid amplification product consistsof 1) a first nucleotide sequence that is continuously complementary toor substantially identical to the first polynucleotide of the firstoligonucleotide, 2) a second nucleotide sequence that is continuouslycomplementary to or substantially identical to the second polynucleotideof the second oligonucleotide, and 3) a spacer sequence comprising 1 to10 bases.

D13.3 The kit of any one of embodiments D1 to D12, further comprisinginstructions for carrying out a method for determining the presence,absence or amount of a target sequence in sample nucleic acid, themethod comprising:

-   -   a) amplifying a target sequence in the sample nucleic acid,        wherein:    -   the target sequence comprises a first strand and a second        strand,    -   the first strand and second strand are complementary to each        other,    -   and the amplifying comprises contacting non-denatured sample        nucleic acid under helicase-free isothermal amplification        conditions with:        -   i) a first oligonucleotide and a second oligonucleotide,            wherein the first oligonucleotide consists of a first            polynucleotide continuously complementary to a sequence in            the first strand, and the second oligonucleotide consists of            a second polynucleotide continuously complementary to a            sequence in the second strand; and        -   ii) at least one component providing a hyperthermophile            polymerase activity, thereby generating a nucleic acid            amplification product, wherein the nucleic acid            amplification product consists of 1) a first nucleotide            sequence that is continuously complementary to or            substantially identical to the first polynucleotide of the            first oligonucleotide, 2) a second nucleotide sequence that            is continuously complementary to or substantially identical            to the second polynucleotide of the second oligonucleotide,            and 3) a spacer sequence comprising 1 to 10 bases, and the            spacer sequence is flanked by the first nucleotide sequence            and the second nucleotide sequence; and    -   b) detecting the nucleic acid amplification product, wherein        detecting the nucleic acid amplification product comprises use        of a real-time detection method and is performed in 10 minutes        or less from the time the sample nucleic acid is contacted with        (a)(i) and (a)(ii), whereby the presence, absence or amount of a        target sequence in sample nucleic acid is determined.

D14. The kit of any one of embodiments D13 to D13.3, wherein the methoddoes not comprise denaturing the sample nucleic acid prior to or duringamplification.

D15. The kit of any one of embodiments D13 to D14, wherein the samplenucleic acid is not contacted with an endonuclease prior to, during, orfollowing amplification.

D16. The kit of any one of embodiments D13 to D15, wherein the samplenucleic acid is unmodified prior to amplification.

D17. The kit of embodiment D16, wherein the unmodified sample nucleicacid is from disrupted cells.

D18. The kit of any one of embodiments D13 to D15, wherein the samplenucleic acid comprises DNA.

D19. The kit of embodiment D18, wherein the sample nucleic acidcomprises genomic DNA.

D20. The kit of any one of embodiments D13 to D15, wherein the samplenucleic acid comprises RNA.

D21. The kit of embodiment D20, wherein the sample nucleic acidcomprises viral RNA.

D22. The kit of embodiment D20, wherein the sample nucleic acidcomprises bacterial RNA.

D23. The kit of any one of embodiments D13 to D22, wherein the samplenucleic acid comprises single-stranded nucleic acid.

D24. The kit of any one of embodiments D13 to D22, wherein the samplenucleic acid comprises double-stranded nucleic acid.

D25. The kit of any one of embodiments D13 to D24, wherein samplenucleic acid is obtained from a subject prior to amplification.

D26. The kit of any one of embodiments D13 to D25, wherein unpurifiedsample nucleic acid is amplified.

D27. The kit of any one of embodiments D13 to D25, wherein purifiedsample nucleic acid is amplified.

D28. The kit of any one of embodiments D13 to D27, wherein the methodfurther comprises purifying sample nucleic acid prior to amplification.

D29. The kit of any one of embodiments D13 to D28, wherein theamplification is performed at a constant temperature of about 55 degreesCelsius to about 75 degrees Celsius.

D30. The kit of any one of embodiments D13 to D28, wherein theamplification is performed at a constant temperature of about 55 degreesCelsius to about 65 degrees Celsius.

D31. The kit of any one of embodiments D13 to D28, wherein theamplification is performed at a constant temperature of about 65 degreesCelsius.

D32. The kit of any one of embodiments D13 to D28, wherein theamplification is performed at a constant temperature of about 60 degreesCelsius.

D33. The kit of any one of embodiments D13 to D32, wherein the nucleicacid amplification product is about 20 to 40 bases long.

D34. The kit of any one of embodiments D13 to D33, wherein the spacersequence comprises 1 to 5 bases.

D35. The kit of any one of embodiments D13 to D34, wherein the spacersequence is not complementary to or identical to the firstpolynucleotide of the first oligonucleotide and is not complementary toor identical to the second polynucleotide of the second oligonucleotide.

D36. The kit of any one of embodiments D13 to D35, wherein the spacersequence is continuously complementary to or substantially identical toa portion of a target sequence in the sample nucleic acid.

D37. The kit of any one of embodiments D13 to D36, wherein detecting thenucleic acid amplification product comprises detection of a fluorescentsignal.

D38. The kit embodiment D37, wherein the fluorescent signal is from amolecular beacon.

D39. The kit of any one of embodiments D13 to D38, wherein the methodfurther comprises contacting the nucleic acid amplification product witha signal generating oligonucleotide that comprises i) a polynucleotidecomplementary to a sequence in the amplification product, and ii) afluorophore and a quencher.

D40. The kit of any one of embodiments D13 to D39, wherein the method isperformed in a single reaction volume.

D41. The kit of any one of embodiments D13 to D40, wherein the method isperformed in a single reaction vessel.

D42. The kit of any one of embodiments D13 to D41, wherein the methodcomprises multiplex amplification.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents. Their citation is not an indication of asearch for relevant disclosures. All statements regarding the date(s) orcontents of the documents is based on available information and is notan admission as to their accuracy or correctness.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent technology has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

What is claimed is:
 1. A method for detecting a target nucleic acidsequence in a sample, the method comprising: (a) amplifying a targetnucleic acid sequence comprising a first strand and a second strandcomplementary to each other in an isothermal amplification condition,wherein the amplifying comprises contacting a non-denatured nucleic acidcomprising the target nucleic acid sequence with: i) a first primer anda second primer, wherein the first primer is capable of hybridizing to asequence of the first strand of the target nucleic acid sequence, andthe second primer is capable to hybridizing to a sequence of the secondstrand of the target nucleic acid sequence; and ii) an enzyme having ahyperthermophile polymerase activity, thereby generating a nucleic acidamplification product at detectable levels within 10 minutes, whereinthe nucleic acid amplification product comprises: (1) the sequence ofthe first primer, and the reverse complement thereof, (2) the sequenceof the second primer, and the reverse complement thereof, and (3) aspacer sequence flanked by (1) the sequence of the first primer and thereverse complement thereof and (2) the sequence of the second primer andthe reverse complement thereof, wherein the spacer sequence is 1 to 10bases long; and wherein the amplifying does not comprise using anyenzyme other than the enzyme having a hyperthermophile polymeraseactivity, and the amplifying does not comprise denaturing thenon-denatured nucleic acid; and (b) detecting the nucleic acidamplification product.
 2. The method of claim 1, wherein step (b)further comprises determining the amount of the non-denatured nucleicacid that comprises the target nucleic acid sequence in the sample. 3.The method of claim 1, wherein the non-denatured nucleic acid is agenomic nucleic acid, a plasmid nucleic acid, a mitochondrial nucleicacid, a cellular nucleic acid, or an extracellular nucleic acid.
 4. Themethod of claim 1, wherein the non-denatured nucleic acid is a bacterialnucleic acid or a viral nucleic acid.
 5. The method of claim 1, whereinthe target nucleic acid sequence is a bacterial nucleic acid sequence ora viral nucleic acid sequence.
 6. The method of claim 1, wherein thenon-denatured nucleic acid is a double-stranded DNA.
 7. The method ofclaim 1, wherein the non-denatured nucleic acid is a product of reversetranscription reaction.
 8. The method of claim 7, wherein thenon-denatured nucleic acid is a product of reverse transcriptionreaction generated from a cellular RNA, a mRNA, a microRNA, a bacterialRNA, or a viral RNA.
 9. The method of claim 1, further comprisinggenerating the non-denatured nucleic acid by a reverse transcriptionreaction before step (a).
 10. The method of claim 1, wherein the enzymehaving a hyperthermophile polymerase activity has a reversetranscriptase activity.
 11. The method of claim 1, wherein the enzymehaving a hyperthermophile polymerase activity has an amino acid sequencethat is at least 90% identical to the amino acid sequence of SEQ ID NO:8or a functional fragment thereof.
 12. The method of claim 1, wherein theenzyme having a hyperthermophile polymerase activity is a polymerasecomprising the amino acid sequence of SEQ ID NO:
 8. 13. The method ofclaim 1, wherein the enzyme having a hyperthermophile polymeraseactivity has 10% or less exonuclease activity compared to an unmodifiedhyperthermophile polymerase.
 14. The method of claim 1, wherein thesample comprises nucleic acids from prokaryotes or eukaryotes.
 15. Themethod of claim 14, wherein the sample comprises nucleic acids from avirus or a bacterium.
 16. The method of claim 1, wherein the method doesnot comprise contacting the non-denatured nucleic acid with asingle-stranded DNA binding protein prior to or during step (a).
 17. Themethod of claim 1, wherein the amplifying the target nucleic acidsequence is performed at a constant temperature between 55 degreesCelsius and 75 degrees Celsius.
 18. The method of claim 17, wherein theamplifying the target nucleic acid sequence is performed at a constanttemperature of 65 degrees Celsius.
 19. The method of claim 1, whereinthe first primer, the second primer, or both is 8 to 16 bases long. 20.The method of claim 1, wherein the nucleic acid amplification product is20 to 40 bases long.
 21. The method of claim 1, wherein the spacersequence comprises a portion of the target nucleic acid sequence. 22.The method of claim 21, wherein the spacer sequence is 1 to 5 baseslong.
 23. The method of claim 1, further comprising contacting thenucleic acid amplification product with a signal-generatingoligonucleotide capable of hybridizing to the amplification product,wherein the single-generating oligonucleotide comprises a fluorophore, aquencher, or both.
 24. The method of claim 1, wherein the detecting thenucleic acid amplification product comprises detecting a fluorescentsignal.
 25. The method of claim 24, wherein the fluorescent signal isfrom a molecular beacon.
 26. The method of claim 1, wherein the methodis performed in a single reaction vessel.
 27. The method of claim 1,wherein the first primer, the second primer, or both comprise one ormore of DNA bases, modified DNA bases, or a combination thereof.